Chromatographic technique for life science researchers
Chromatographic technique for life science researchers Presented by Dr. B. Victor., Ph.D.,email : email@example.com blog : bonvictor.blogspot.com
Presentation outlinePresentation Science of chromatographyoutline Meaning, origin, scope and definition. Principles, Rf value and basic categories Classification of chromatography Research applications Research choice of chromatographic methods Significance of chromatography.
Science of chromatography What is chromatography? • Chromatography is the science of separation techniques . • The technique is used to fractionate mixture of gases, liquids or dissolved solids.
Meaning of chromatography Color writing • The name chromatography (Greek: chroma means color and graphein means writing)literally means writing in color. • In other words writing out the ‘signature ‘ of a mixture in color.
Origin of chromatographic methods The Russian botanist Mikhail S. Tswett (1872- 1919) found that pigment composition became separated when plantMikhail S. Tswett pigment (chlorophyll)(1872-1919) together with petroleum ether went through calcium carbonate layer.
Scope of chromatography Statement of Tswett (1906) • Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system.
Definitions of chromatography Definition -1 • Chromatography is a physical separation technique used to separate macromolecules based on their size, shape or charge (Heftmann 1992 ). Definition -2 • The separation of components of a mixture based on differential distribution between a mobile and stationary phase.
Science of chromatography Chromatography is a Chromatographic – like dynamic separation systemseparation processes occur which partitions chemical in nature –for e.g. substances between twomigration of water through phases (Biphasic system)-asoils results in purification stationary phase (SP) and a of water. mobile phase(MP). Chromatography is a science which studies the Chromatography is a separation of molecules flexible yet powerful based on differences in analytical procedure. their structure and / or composition.
Stationary and Mobile phases in chromatography Biphasic system Mobile phase can be Stationary phase a gas or a liquid which consists of small solid carries the particles with micro components of a porous surface mixture. The rate of Stationary phase movement of a given More solubleThe stationary phase may be packed up in component of a substances travel may be a solid or a column or spread mixture depends on more slowly downliquid supported on a as a layer or the degree of the column than the solid or a gel. distributed as a film. solubility in the less soluble. solvent system.
Basic categories of chromatographic techniques Based on Separation Based on Stationary mobile phase techniques phase Liquid Liquid-solid Solid chromatography Adsorption Gas chromatography Gas-solid chromatographyChromatographic methods Liquid Liquid-liquid Liquid chromatography Partition chromatography Gas Gas-liquid chromatography
Principle of surface adsorptionAdsorption is the property holding molecules at the surfaceof a solid or liquid molecules. Solute Adsorption coefficient =degree of adsorption molecules Molecule Interaction specific Adsorption forces between based on Van der vaals forces Electrostatic forces Solvent Molecular Polar forces chemisorption molecules structure
Surface adsorbents= finely divided porous solids Choice of Determinants of adsorbents Adsorption capacity Silica gel Alumina Surface area Powdered sugar Chemical composition Activated charcoal Magnesium carbonate Magnesium silicate Geometrical arrangements of the Calcium carbonate groups /atoms Sodium carbonate
Basic characteristics of adsorbents They are micro They offer porous solid They never react mechanical stability particles with chemically with during columnregular or irregular solutes or solvents packing surfaces They retain small They are amount soluble catalytically inactive components
Requirements Insoluble in mobile phaseof an ideal Non-reactive to solutes.adsorbent Colorless when separating colored mixture. Choice various particle sizes.
kinds of Adsorption chromatography Adsorption column chromatography Adsorption Thin layerchromatography chromatography Ion exchange chromatography
Principle of partition Partition is the relative(differential) solubility of Partition coefficient is components of a the degree of solubilitymixture in two or more immiscible solvents. More soluble substances travel more slowly down the column than the less soluble.
Basic characteristics of solvent systems The composition of the solvent system should be stable through out the development. The developing solvent should move at a relatively slow rate. The choice of the solvent should be one in which the components have small but definite solubility.
Principle of separationDistribution co-efficient K :During separation, there is a relationship betweenthe amount of solute retained by one phase inrelation to another phase.In partition method the distribution co-efficient ispartition co-efficient i.e. solubility of solute in thestationary and mobile phases.In adsorption method , the distribution co-efficientis adsorption co-efficient i.e. the amount soluteadsorbed by the stationary and mobile phases.
Principle of separation Solvent flow A B A B A B AB Stationary organic phase Wall of columnSample Component A Component BMixture
Rf value : relative front The relation of the distance travelled by compound to that of the solvent front is called Rf value. Rf value = Distance travelled by the solute ------------------------------------------ Distance travelled by the solvent Parameter influencing Rf value Temperature, Solvent system Direction of flow, Type of paper.
Kinds of chromatography Gas-solid Chromatography Gas mobile GSC phase Gas-solid Chromatography GLC Column chromatography Liquid-solid Chromatography LSC Liquid-liquid Liquid mobile Chromatography phase LLCChromatographic techniques Bonded Phase Chromatography Paper chromatography Liquid mobile Planar chromatography phase Thin layer chromatography
Chromatographic techniques based solvent system Uni- Two dimensional dimensional Single Solvent Two System Solvent systems One directional Two directions solvent flow
Modifications of chromatographic methods -1Multiple development Continuous development The paper is developed The chromatogram is with the same solvent developed in the same system several times in solvent system in the the same direction. same direction continuously for a long time.
Modifications of chromatographic methods -2Bidirectional Reverse phasechromatography chromatography The chromatogram is For the separation of first developed in one hydrophobic solvent system in one compounds like fatty direction. acids, the filter paper is It is again developed in treated with lipophilic a second solvent compounds (silicon system in direction greese, kerosene oil), perpendicular to first while the mobile phase run. is hydrophilic in nature.
Modifications of chromatographic methods-3 One way or Linear flow of solvent • Column, strips, sheets. • Thin layer of sorbent Two –way or transverse solvent flow • sheets • Thin layer Radial or horizontal Solvent flow • Circular paper discs • Paper Sheet
Types of chromatographic methods Based on Based on solvent quantity direction Micro-scale Up-ward Laboratory assays ascending Paper, TLC Medium scale Down ward Analytical assays descending Column flow Large scale Horizontal Commercial Transverse GLC, GSC radial
Chromatographic development : principles of separation-1Elution development Gradient elution The components of the A gradual change in mixture are separated composition of the eluting solvent is used to achieve into zones by the separation of compounds passage of one or more of widely varying affinities solvents through the for the stationary phase. column. The solvent composition This technique is most gradient may be linear with widely used in GC, increasing or decreasing concentration, pH, polarity GLC, LLC and LSC. or ionic strength.
Chromatographic development : principles of separation-2Frontal analysis Displacement analysis No solvent is used for The components in the mixture are adsorbed on irrigation. the column. The solution itself is The irrigation of the added continuously. column is carried out with the solution of another substance, having a higher preferential adsorption on the column than that of the components of the mixture sought.
Research applications of chromatographic methodsAnalytical applications Preparative applications To determine the To purify and collect chemical composition one or more of a biological sample. components of a biological sample.
Research choice of chromatographic methods SubstancesOf similar chemical type • Partition chromatography Substances of different chemical type • Adsorption chromatography Gases and volatile substances • Gas chromatography Ionic and inorganic • Ion exchange chromatography substances • Column or paper or thin layerBiological materials and Compounds of relative • Gel chromatography High molecular mass
Protocol of chromatography methodsColumn chromatography Paper chromatography A mixture of components The paper adsorbs dissolved in a solvent is water from the poured over a column of solid adsorbent . atmosphere of the The column is eluted developing with the same or a chromatogram. different solvent. The water is the The stationary phase is stationary phase. solid. The eluting solvent is The mobile phase (the eluent) is liquid. the mobile phase.
Significance of chromatographic methodsThey serve to resolve Very small quantities and identify the of substances could The equipment is very separated be analyzed simple except HPLC components of a qualitatively and mixture. quantitatively. No special skill is The results are required for remarkably performing the reproducible. method
Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’ s College, Palayamkottai, India-627001. He was the dean of sciences, IQAC coordinator and assistant controller of examinations. He has more than 32 years of teaching and research experience He has taught a diversity of courses and guided 12 Ph.D scholars. Send your comments to : firstname.lastname@example.org