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Page 1: Biochemical instrumental analysis - 11 Dr. Maha Al-Sedik 2015 CLS 332.

Biochemical instrumental analysis - 11

Dr. Maha Al-Sedik2015

CLS 332

Page 2: Biochemical instrumental analysis - 11 Dr. Maha Al-Sedik 2015 CLS 332.

Ion exchange chromatography

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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

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Cations are attached to the stationary phase, so it attract anions.

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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.

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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).

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Anion exchanger chromatography

Cation exchanger chromatography

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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.

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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.

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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.

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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.

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Ion exchange chromatography can be subdivided to:

1- Cation exchange chromatography.

2- Anion exchange chromatography.

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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.

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Chromatography in which the mobile phase is a liquid.

The stationary phase is usually a solid or a high viscous liquid.

Liquid Chromatography

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Liquid chromatography

Liquid solid chromatography

Liquid liquid chromatography

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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) .

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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.

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In principle, LC and HPLC work the same way

except the speed and ease of operation of HPLC is

vastly superior.

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High performance liquid chromatography

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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

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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).

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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.

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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.

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Column:

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Detector:

The detector can detect the individual molecules that

come out (elute) from the column.

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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).

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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.

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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.

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