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OutlineDefinition of ChromatographyHistory of Chromatography
Chromatography terms
Classification of Chromatography
Chromatograph
Applications of Chromatography
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Definition
Is the collective term for a family of laboratory techniques
for the separation of mixtures
Involves passing a mixture which contains the analyte
through a stationary phase, which separates it from other
molecules in the mixture and allows it to be isolated
SeparateAnalyze Identify
Purify
QuantifyComponentsMixture
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Chromatography Terms
The analyte is the substance that is to be separated
during chromatography
Stationary phase: Phase that stays in place inside thecolumn. Can be a particular solid or gel-based packing (LC)
or a highly viscous liquid coated on the inside of the
column (GC)
Mobile phase: Solvent moving through the column,
either a liquid in LC or gas in GC
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Eluent: Fluid entering a column
Eluate: Fluid exiting the column
Elution: The process of passing the mobile phase
through the column.
Chromatogram: Graph showing detector response
as a function of a time.
Flow rate: How much mobile phase passed /
minute (ml/min).
Linear velocity: Distance passed by mobile phase
per 1min in the column (cm/min)
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Chromatography
Column Planar Thin layer
Paper
physical state ofmobile
phase
GasLiquid
Supercritical
fluid
Separation mechanism
Size
exclusion
Ion
exchange
Classification
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Column Chromatography
The stationary phase (column
packing) in the column is very polar
One starts will a less polar solvent to
remove
the less polar compounds, and then
you slowly
Increase the polarity of the solvent to
remove the more polar compounds
Figure 1. column chrotography .
solvent
column
Bands of
separatedconsistuents
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Thin LayerChromatography
Here the mobile phase is a liquid
Flowing past a thin layer of powder on a solid
support
Substances that are less attracted to the solid or are
more soluble in the liquid move faster
And so move further up the plate by the time that the
process has been stopped by taking the plate out of
the liquid. - larger Rf
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Figure 2. principles in TLC.
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PaperChromatography
Figure 3. Paper chromatography jar
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Size exclusion ChromatographyGEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of
molecules based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are,
therefore, retained longer than large molecules.
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Separation in Ion-exchange Chromatography is based on
the competition of different ionic compounds of the sample
for the active sites on the ion-exchange resin (column-
packing).
Ion- exchange Chromatography
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Liquid chromatography (LC) is a separation technique in
which the mobile phase is a liquid. Liquid
chromatography can be carried out either in a column or
a plane
Liquid Chromatography
Present day, use high
performance liquid
chromatography (HPLC)
Figure 4 . HPLC 1525
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Gas Chromatography
Principals of SeparationColumn is selected, packed with Liquid Phase, and installed.
Sample injected with microliter syringe into the injection
port where it is vaporized and mixed into the Carrier Gas
stream (helium, nitrogen, argon).
Sample vapor becomes partitioned between Moving Gas
Phase and Stationary Liquid Phase
The time the different compounds in the sample spend in
the Vapor Phase is a function of their Vapor Pressure.
The more volatile (Low Boiling Point / Higher Vapor
Pressure) compounds arrive at the end of the column first
and pass into the detector
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Chromatograph
Figure 5. chromatograph
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Figure 6. chromatographic peak is fronting
Figure 7. chromatographic peak is tailing
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Applications
Figure 6. Gas chromatographyFigure 5. HPLC
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Chromatography is used by scientists to:
Analyze examine a mixture, its components,and their relations to one another
Identify determine the identity of a mixture orcomponents based on known components
Purify separate components in order to isolate
one of interest for further study
Quantify determine the amount of the a mixtureand/or the components present in the samplecxxc
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Pharmaceutical Company determine amount ofeach chemical found in new product
Hospital detect blood or alcohol levels in apatients blood stream
Law Enforcement to compare a sample found at
a crime scene to samples from suspects
Environmental Agency determine the level ofpollutants in the water supply
Manufacturing Plant to purify a chemical neededto make a product
So on
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References
http://en.wikipedia.org/wiki/Chromatography
www.chromatography-online.org
www.docjax.com/.../index.shtml?...thin%20layer%20 chomatography
www.ebookpdf.net/__chomatography-free-book_ebook_.html
www.waters.com/.../library.htm...