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

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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.