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Biochemical instrumental analysis - 11
Dr. Maha Al-Sedik2015
CLS 332
Ion exchange chromatography
Principle:
Positively or negatively charged groups are immobilized on a
stationary phase.
Ion Exchange Chromatography is based on the binding of proteins
to the opposite charge.
Ion exchange chromatography
Cations are attached to the stationary phase, so it attract anions.
Used for almost any kind of charged molecules --- large proteins,
small nucleotides and amino acids.
Mobile phase: liquids , pH must be controlled .
opposite charged solute ions attracted to the stationary phase by
electrostatic force.
Stationary phase: resin ( solid ) is used to covalently attach anions
or cations onto it.
Types of resin:
A- Cation exchanger resin: Has negatively charged groups in the resin
and so it will attract positively charged (cation )molecules.
B- Anion exchanger resin: Has positively charged groups in the resin
and so it will attract negatively charged molecules ( Anion).
Anion exchanger chromatography
Cation exchanger chromatography
Most ion exchange experiments are performed in five main stages:
The first stage:
Is equilibration stage in which the ion exchanger is brought to a
starting state by attaching weak opposite charge to the ionic group
immobilized on the gel.
The second stage:
Is sample application and adsorption, in which solute molecules
carrying the appropriate charge displace counter-ions and bind
reversibly to the gel. Unbound substances can be washed out from
the exchanger bed using buffer.
In the third stage: • Is starting of elution.
• Samples are removed from the column by changing to elution
conditions unfavorable for ionic bonding of the solute molecules.
• This normally involves changing its pH.
The fourth and fifth stages:
Stage of end of elution and re-equilibration.
The removal of substances not eluted under the previous
experimental conditions and
re-equilibration at the starting conditions for the next purification.
Ion exchange chromatography can be subdivided to:
1- Cation exchange chromatography.
2- Anion exchange chromatography.
Applications:
1- Separation of a mixture of amino acids.
2- Fractionation of plasma proteins, hemoglobin and hormones.
3- Remove substance that may interfere reaction in any tests.
Chromatography in which the mobile phase is a liquid.
The stationary phase is usually a solid or a high viscous liquid.
Liquid Chromatography
Liquid chromatography
Liquid solid chromatography
Liquid liquid chromatography
Thin layer chromatography and column chromatography are
examples of liquid chromatography.
The main problem with liquid chromatography is time required
for analysis.
To improve the performance of liquid chromatography, We
started to do liquid chromatography under high pressure ( high
performance liquid chromatography) .
Mobile Phase:
Water “Ultrapure water” can be used.
Commercial “distilled water for HPLC” is also acceptable.
Organic Solvent HPLC grade solvent can be used.
Special grade solvent is acceptable depending on the detection
conditions.
In principle, LC and HPLC work the same way
except the speed and ease of operation of HPLC is
vastly superior.
High performance liquid chromatography
HPLC is a separation technique where individual components of
the sample are moved down the column ( stationary phase)
with a liquid (mobile phase) forced through the column by
high pressure delivered by a pump.
Definition of high performance liquid chromatography
components:
Pump:
The role of the pump is to force the mobile phase through the
liquid chromatograph at a specific flow rate, expressed in milliliters
per min (mL/min).
Injector:
The injector serves to introduce the liquid sample into the
flow stream of the mobile phase.
Typical sample volumes are 5 – 20 microliters (μL).
The injector must also be able to withstand the high
pressures of the liquid system.
Column:
Considered the “heart of the chromatograph”.
The pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within the
chromatograph.
Column:
Detector:
The detector can detect the individual molecules that
come out (elute) from the column.
Computer:
Frequently called the data system.
The computer controls all the modules of the HPLC instrument.
It takes the signal from the detector and uses it to determine
the time of elution (retention time) of the sample components
(qualitative analysis) and the amount of sample (quantitative
analysis).
Clinical applications :
Separation and analysis of liquid non-volatile compounds.
If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in
gasoline, etc.), gas chromatography is a better separation technique.
Clinical applications :
Qualitative analysis: The components of a mixture can be identified by direct comparison
of their retention time with that obtained for reference compounds.
Quantitative analysis:An important advantage of liquid chromatography is its ability to
separate and quantitate the multiple components of a mixture e.g.
drugs and allied substances.