Introduction to Instrumental Analysis - Chromatography

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What is chromatography? The principle of chromatography. Session Objectives: What is chromatography? The principle of chromatography. Chromatography is used in. Classification of Chromatographic Methods. Column chromatography. Classification of column chromatography. Planar chromatography.

Transcript of Introduction to Instrumental Analysis - Chromatography

Introduction to Instrumental Analysis - Chromatography
Clinical Analytical Chemistry CLS 231 Introduction to Instrumental Analysis - Chromatography Lecture 12 Lecturer: Amal Abu-Mostafa What is chromatography? The principle of chromatography.
Session Objectives: What is chromatography? The principle of chromatography. Chromatography is used in. Classification of Chromatographic Methods. Column chromatography. Classification ofcolumn chromatography. Planar chromatography. What is chromatography?
Chromatography (from Greek wordchromosfor colour) is the collective term for a family of laboratory techniques for the separation of mixtures. It is a technique used to separate and identify the components of a mixture. Mobile phase Stationary phase Column The principle of chromatography
It involves passing a mixture which contains the analyte through a stationary phase, which separates it from other molecules in the mixture and allows it to be isolated. During the process of chromatography, the components of a sample distribute themselves between the mobile phase (which may be a gas, a liquid, or a supercritical fluid.) and stationary phase (, which is fixed in place in a column or on a solid surface). Molecules that spend most of their time in the mobile phase are carried alongfaster. Chromatography is used in:
Chromatography is a widely used method that allows the separation, identification, and determination of the chemical components in complex mixtures (such as proteins,vitamins,sub cellular compartments. etc.). We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances. Classification of Chromatographic Methods
Chromatographic methods can be categorized in two ways. The first according to the shape of the stationary phase: 1) Column chromatography 2) Planar chromatography Classification of Chromatographic Methods
1) Column chromatography In column chromatography, the stationary phase is held in a narrow tube through which the mobile phase moves under the influence of gravity or pressure. Mobile phase Stationary phase Column General Theory of Column Chromatography
A typical column chromatography experiment is outlined in Figure 12.4. General Theory of Column Chromatography
The sample is introduced at the top of the column as a narrow band. Ideally, the solutes initial concentration profile is rectangular (Figure 12.5a). As the sample moves down the column the solutes begin to separate, and the individual solute bands begin to broaden and develop a Gaussian profile (Figures 12.5b,c). If the strength of each solutes interaction with the stationary phase is sufficiently different, then the solutes separate into individual bands (Figure 12.5d). The progress of a chromatographic separation is monitored with a suitable detector situated at the end of the column. A plot of the detectors signal as a function of time or volume of eluted mobile phase is known as a chromatogram (Figure 12.6) and consists of a peak for each of the separated solute bands. A more fundamental classification of Column chromatographic methods is one based upon the types of mobile phases: (1) liquid chromatography (2) gas chromatography (3) supercritical-fluid chromatography. The mobile phases in the three techniques are liquids, gases, and supercritical fluids respectively. Classification of column chromatography
Liquid chromatography includes the following methods: (a) Ion-exchange chromatography (b) Size-exclusion chromatography (c) Affinity chromatography (d) HPLC, or high-performance liquid chromatography For example three of the column chromatographic methods are used in protein purification (a), (b) and (c). (a) Ion-exchange chromatography Ion-exchange chromatography A form of liquid chromatography in which the stationary phase is an ion- exchange resin. (a) Ion-exchange chromatography (a) Ion-exchange chromatography
In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups. The counterions to these fixed charges are mobile and can be displaced by ions that compete more favorably for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation exchangers; weak acid cation exchangers; strong base anion exchangers; and weak base anion exchangers. (b) Size-exclusion chromatography
Size-exclusion chromatography: A form of liquid chromatography in which the stationary phase is a porous material and in which separations are based on the size of the solutes. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solutes ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. (b) Size-exclusion chromatography (c) Affinity chromatography
The stationary phase is a group specific liquid bonded to a solid surface. Affinity chromatography Separates proteins by their binding specificities. The proteins retained on the column are those that bindspecifically to a ligand cross-linked to the beads. (In biochemistry, theterm ligand is used to refer to a group or molecule that binds to amacromolecule such as a protein.) After proteins that do not bind tothe ligand are washed through the column, the bound protein ofparticular interest is eluted (washed out of the column) by a solutioncontaining free ligand. (c) Affinity chromatography (d) High-performance liquid chromatography HPLC:
High-performance liquid chromatography: A chromatographic technique in which the mobile phase is a liquid also. Classification of Chromatographic Methods
2) Planar chromatography In planar chromatography, the stationary phase is supported on a flat plate or in the interstices of a paper; here, the mobile phase moves through the stationary phase by capillary action or under the influence of gravity. Example on Planner Chromatography
Paper Chromatography Can be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances Thin-Layer Chromatography Uses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances. Thank you