B IOCHEMICAL 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.

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).  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.