Transcript Online prac of HPLC

Chromatography- TLC &
HPLC
By Chloe
Holmberg
TLC- Thin Layer Chromatography
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
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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.
TLC-Interpreting TLC
chromatograms
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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.
TLC- Limitations and advantages
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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.
TLC- Elutropic series
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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.
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
Bibliography- TLC
http://www.chemilp.net/labTechniques/ElutropicSeries.ht
m
 http://www.chemguide.co.uk/analysis/chr
omatography/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.
Pump &
solvent
reservoir
HPLC
Column
Sample
injection point
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.
HPLC- Apparatus
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The column
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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.
HPLC- Apparatus
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The solvent (liquid mobile phase)
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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.
HPLC- Apparatus
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The detector
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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.
HPLC- Interpreting HPLC
chromatograms
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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.
HPLC- Interpreting HPLC
chromatograms
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
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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
bibliography- HPLC
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http://www.chemguide.co.uk/analysis/chromatography/hplc.html#top
 http://www.google.com.au/imgres?q=HPLC+chromatograms+and+calibr
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