BIOCHEMICAL INSTRUMENTAL
ANALYSIS-11
Dr. Maha Al-Sedik
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.
Liquid Chromatography
Chromatography in which the mobile phase is a
liquid.
The stationary phase is usually a solid or a high
viscous liquid.
In general, it is possible to analyze any substance
that can be stably dissolved in the mobile phase.
Liq
uid
ch
rom
ato
gra
ph
yLiquid 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).
During the chromatographic experiment, a pump can
deliver a constant mobile phase composition.
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
column’s stationary phase separates the sample
components of interest using various physical and
chemical parameters.
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.
A detector serves to measure the amount of
those molecules so that the chemist can
quantitatively analyze the sample
components.
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.