Chromatographic Analyzers

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What is Chromatography?

 Chromatography is a method used by scientists for separating organic


and inorganic compounds so that they can be analyzed and studied. By
analyzing a compound, a scientist can figure out what makes up that
compound.

 Chromatography is a great physical method for observing mixtures and


solvents.
Brief History
 The word chromatography means "color writing" which is a way that a
chemist can test liquid mixtures. While studying the coloring materials
in plant life, a Russian botanist invented chromatography in 1903. His
name was M.S. Tswett.

 Chromatography is such an important technique that two nobel prizes


have been awarded to chromatographers. Over 60% of chemical
analysis worldwide is currently done with chromatography or a
variation thereon.
How it works?
 Stationary phase is the phase that doesn't move and the mobile phase is
the phase that does move.

 Mobile phase moves through the stationary phase picking up the


compounds to be tested.

 As the mobile phase continues to travel through the stationary phase it


takes the compounds with it. At different points in the stationary phase
the different components of the compound are going to be absorbed
and are going to stop moving with the mobile phase. This is how the
results of any chromatography are gotten, from the point at which the
different components of the compound stop moving and separate from
the other components.
What is the Retention Factor, Rf ?
 It is a quantitative indication of how far a particular compound travels
in a particular solvent. The Rf value is a good indicator of whether an
unknown compound and a known compound are similar, if not
identical.
What is the Retention Factor, Rf ?
 The retention factor, Rf, is defined as
Rf = D1 / D2

Rf = distance the solute (D1) moves divided by the


distance traveled by the solvent front (D2)

D1 = distance that color traveled, measured from


center of the band of color to the point
where the food color was applied

D2 = total distance that solvent traveled


The Different Types of Chromatography:
 Liquid Chromatography
 Gas Chromatography
 Thin-Layer Chromatography
 Paper Chromatography
The Different Types of Chromatography:
Type of Chromatography Applications in the Real World Why and What is it

Used to analyze metal ions and


organic compounds in solutions.
test water samples to look for
Liquid Chromatography It uses liquids which may
pollution
incorporate hydrophilic, insoluble
molecules.

detect bombs in airports, identify Used to analyze volatile gases.


and quantify such drugs as Helium is used to move the
Gas Chromatography
alcohol, used in forensics to gaseous mixture through a
compare fibers found on a victim column of absorbent material.
The Different Types of Chromatography:
Type of Chromatography Applications in the Real World Why and What is it

detecting pesticide or insecticide Uses an absorbent material on flat


residues in food, also used in glass plates. This is a simple and
Thin-Layer Chromatography
forensics to analyze the dye rapid method to check the purity
composition of fibers of the organic compound.

The most common type of


separating amino acids and chromatography. The paper is the
anions, RNA fingerprinting, stationary phase. This uses
Paper Chromatography
separating and testing histamines, capillary action to pull the solutes
antibiotics up through the paper and
separate the solutes.
High Performance Liquid
Chromatography (HPLC)
 A highly improved form of column chromatography with highly
automated and extremely sensitive detection methods

 Instead of a solvent being allowed to drip through a column under


gravity, it is forced through under high pressures of up to 400
atmospheres which makes it much faster

 Allows the use of very much smaller particle size for the column
packing material which gives a much greater surface area for
interactions between the stationary phase and the molecules flowing
past it resulting to a better separation of the components of the mixture
How does it work?
 A reservoir holds the solvent (mobile phase) while a high-pressure
pump is used to generate and meter a specified flow rate (typically in
mL/min) of mobile phase. On the other hand, an injector inject the
sample into the continuously flowing mobile phase stream that carries
the sample into the HPLC column. The column contains the
chromatographic packing material needed to effect the separation.
This packing material is called the stationary phase because it is held
in place by the column hardware. Also, a detector is needed to
detect the separated compound bands as they elute from the HPLC
column. Afterwards, the mobile phase exits the detector and can be
sent to waste, or collected, as desired. Additionally, the detector is
wired to the computer data station which records the electrical signal
needed to generate the chromatogram on its display and to identify
and quantitate the concentration of the sample constituents.
2 Basic Elution Modes in HPLC
 Isocratic Elution - the mobile phase (either a pure solvent
or a mixture) remains the same throughout the run.

 Gradient Elution - the mobile phase composition changes


during the separation and is useful for samples that
contain compounds that span a wide range of
chromatographic polarity because as the separation
proceeds, the elution strength of the mobile phase is
increased to elute the more strongly retained sample
components.
HPLC Operation
Detector and Chromatogram
 The detector contains a flow cell that detects each separated
compound band against a background of mobile phase. An
appropriate detector has the ability to sense the presence of a
compound and send its corresponding electrical signal to a
computer data station. A choice is made among many different types
of detectors, depending upon the characteristics and concentrations
of the compounds that need to be separated and analyzed.

 On the other hand, a chromatogram is a representation of the


separation that has chromatographically occurred in the HPLC
system. A series of peaks rising from a baseline is drawn on a time
axis. Each peak represents the detector response for a different
compound. The chromatogram is plotted by the computer data
station.
Identifying and Quantitating Compounds
 From the figure, three dye compounds are represented by three
peaks separated in time in the chromatogram. Each elutes at a
specific location, measured by the elapsed time between the
moment of injection [time zero] and the time when the peak
maximum elutes. By comparing each peak’s retention time with
that of injected reference standards in the same chromatographic
system [same mobile and stationary phase], a chromatographer
may be able to identify each compound.

 Under this liquid chromatography system conditions, the


analyte, acrylamide, would be separated and elute from the
column at 2.85 minutes [retention time]. Whenever a new
sample, which happened to contain acrylamide, was injected
into the LC system under the same conditions, a peak would be
present at 2.85 minutes
Identifying and Quantitating Compounds
 From the figure, chromatograms for Samples A and B, on the same
time scale, are stacked one above the other. The same volume of
sample was injected in both runs. Both chromatograms display a
peak at a retention time of 2.85 minutes, indicating that each
sample contains acrylamide. However, Sample A displays a much
bigger peak for acrylamide. The area under a peak [peak area
count] is a measure of the concentration of the compound it
represents. This area value is integrated and calculated
automatically by the computer data station. In this example, the
peak for acrylamide in Sample A has 10 times the area of that for
Sample B. Using reference standards, it can be determined that
Sample A contains 10 picograms of acrylamide, which is ten times
the amount in Sample B [1 picogram].
Advantages and Disadvantages
of Using HPLC
Advantages Disadvantages
 Extremely quick and efficient (process  Costly
can be completed in roughly 10-30  Complex to troubleshoot problems or
mins) to develop new methods
 High elution resolution  Low sensitivity to certain compounds
 Accurate and highly reproducible
 Can be performed with minimal
training
 Versatile and extremely precise in
identifying and quantifying chemical
components
Materials of Construction:
o For the tubing:
• Stainless steel – the most popular; gives high P capabilities
• Glass – mostly for biomolecules
• PEEK (polyetheretherketone) – for polymers; valued for its biocompatibility
and is chemically inert to most solvents

o For the packing material:


-prepared from Silica particle, Alumina particle and Ion Exchange Resin

o For the column’s end


-porous plug of stainless steel or Teflon are used to retain the packing material
Applications of HPLC:
 Water purification
 Ion-exchange chromatography of proteins
 High-pH anion-exchange chromatography of carbohydrates and
oligosaccharides
 Pharmaceutical development of antibiotic products
 Clinical diagnosis of diseases and disorders
 Isolation and identification of mixture of components
 Biopharmaceutical and Pharmacokinetic studies

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