Chromatography-Edited PDF

UNIT III: INSTRUMENTAL METHODS OF ANALYSIS CHROMATOGRAPHY Instructor Dr. Najbul Hoque  Chromatography Chromatography Chromatography is taken to refer generally to the separation of components in a sample by distribution of the components between two...

UNIT III: INSTRUMENTAL METHODS OF ANALYSIS CHROMATOGRAPHY Instructor Dr. Najbul Hoque  Chromatography Chromatography Chromatography is taken to refer generally to the separation of components in a sample by distribution of the components between two phases—one that is stationary and one that moves, usually (but not necessarily) in a column. Russian botanist, Mikhail Tswett, invented adsorption chromatography during his research on plant pigments. He separated different colored chlorophyll and carotenoid pigments of leaves by passing an extract of the leaves through a column of calcium carbonate, alumina. He coined the term chromatography in a 1906 publication, from the Greek words chroma meaning “color” and graphos meaning “to write.”  Chromatography Definition IUPAC has drafted a recommended definition of chromatography: “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase), while the other (the mobile phase) moves in a definite direction”  Chromatography Apparatus in Chromatography  Chromatography Various Terminologies Stationary Phase The stationary phase in chromatography is a phase that is fixed in place either in a column or on a planar surface. Mobile Phase The mobile phase in chromatography is a phase that moves over or through the stationary phase carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid. Elua te The mobile phase that exits the column is termed the eluate.  Chromatography Various Terminologies Elue An nt eluent is a solvent used to carry the components of a mixture through a stationary phase. Adsorbat e A material that is capable of being adsorbed is known as adsorbate. Example- Oxygen molecule adsorbed on cobalt. Here oxygen is adsorbate. Retention time it is specific time required for a particular analyte to pass through the system Chromatogra mA chromatogram is a plot of some function of solute concentration versus elution time or elution volume.  Chromatography Principle of Chromatography Chromatography is a separation method where the analyte is contained within a liquid or gaseous mobile phase, which is pumped through a stationary phase. Usually, one phase is hydrophilic and the other lipophilic. The components of the analyte interact differently with these two phases. Depending on the polarity they spend more or less time interacting with the stationary phase and are thus retarded to a greater or lesser extent. This leads to the separation of the different components present in the sample.  Chromatography Advantages of chromatography Can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid. Can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Exact quantitative analysis is done even from trace compounds. Small material consumption. Analyses of several compound can be done during one run. Chromatography is a fast analysis method.  Chromatography Classification of Chromatographic Techniques Chromatographic processes can be classified according to the type of equilibration process involved, which is governed by the type of stationary phase. Various bases of equilibration are: (1) adsorption, (2) partition, (3) ion exchange, and (4) size dependent pore penetration. More often than not, solute stationary-phase–mobile phase interactions are governed by a combination of such processes.  Chromatography adsorption Chromatography Adsorption, or liquid-solid, chromatography is the classic form of liquid chromatography. In this chromatographic mode, the stationary phase is a finely divided solid to maximize the surface area. The stationary phase (adsorbent) is chosen to permit differential interaction with the components of the sample to be resolved. The components distribute between the two phases through a combination of sorption and desorption processes. Thin-layer chromatography (TLC) is a special example of adsorption chromatography in which the stationary phase is planar, in the form of a solid supported on an inert plate, and the mobile phase is a liquid.  Chromatography adsorption Chromatography: Column Chromatography The basic principle is selective adsorption. The long column is used to separate the mixture of substances. The solvent system that passes through the column is known as eluent. The process is known as elution. The substance having higher adsorbent capacity retains in upper part of column. The substance having less adsorbent capacity retains in lower part of column. As a result substances retain in different parts of column and separated partially. The solvent is passed again to have better separation.  Chromatography Column Chromatography: pictorial view  Chromatography Column Chromatography: requirements  Chromatography Column Chromatography: requirements  Chromatography Column Chromatography  Chromatography Column Chromatography  Chromatography adsorption Chromatography: Principle The mixture of gas or liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at different rates. Adsorbent – A substance which is generally porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces is called adsorbent. Some commonly used adsorbents are Silica gel, hydrated silica gel, alumina etc.  Chromatography adsorption Chromatography: Principle Caption  Chromatography adsorption Chromatography: Experiment Before starting with the adsorption chromatography experiment let us understand the two types of phases and the types of forces involved during the mixture separation process. Stationary phase – Adsorbent is the stationary phase in adsorption chromatography. The forces involved help to remove solutes from the adsorbent so that they can move with the mobile phase. Mobile phase – Either a liquid or a gas is used as a mobile phase in adsorption chromatography. Forces involved help to remove solutes from the adsorbent so that they can move with the mobile phase. When a liquid is used as a mobile phase it is called LSC (Liquid-Solid Chromatography). When a gas is used as a mobile phase it is called GSC (Gas-Solid Chromatography).  Chromatography adsorption Chromatography: TLC  Chromatography adsorption Chromatography: TLC  Chromatography adsorption Chromatography: TLC  Chromatography adsorption Chromatography  Chromatography adsorption Chromatography: Application In monitoring the progress of reactions Identify compounds present in a given mixture Adsorption chromatography is used for separation of amino acids. It is used in the identification of carbohydrates, fats and fatty acids. Identification of medicinal plants and their constituents  Chromatography Classification of Chromatographic Techniques Chromatography Chromatography