Chromatography and Separation Techniques PDF

Chromatography and Separation Techniques Assoc. Prof. Sameh Younis Associate Professor of Pharmaceutical Analytical Chemistry Week 1  GC Gas Chromatography HPLC High Performance Liquid...

Chromatography and Separation Techniques Assoc. Prof. Sameh Younis Associate Professor of Pharmaceutical Analytical Chemistry Week 1  GC Gas Chromatography HPLC High Performance Liquid Chromatography 2 3  What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, purify, identify, and/or quantify the mixture or components. Analyze Separate Identify Purify Quantify Mixture Components 4 Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample 5  Definition of Chromatography Greek words---- Khroma: color, Graphein: to write Separation, identification and quantification Russian botanist (Tswett)---- father of chromatography petroleum ether chalk *- Stationary Phase (column) (Fixed)(Retarder) *- Mobile Phase (M.P.) (Movable)(Carrier). Different affinities------ different migration rates High affinity to S.P----- late elution ,moves slowly with MP Low affinity to S.P-----early elution ,moves rapidly with MP  Definition of Chromatography Explanation: Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase carries the individual components with a certain speed through the stationary phase, depending on their attraction to both of the phases. 7 Illustration of Chromatography Stationary Phase Separation Mobile Mixture Phase Components Affinity to Mobile Components Affinity to Stationary Phase Phase Blue ---------------- Insoluble in Mobile Phase Black ✓✓✓✓✓✓ ✓✓ Red ✓✓ ✓✓✓✓✓ Yellow ✓ ✓✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 8  Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects “forensic medicine authority” “FMA” Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product 9 Classification of chromatography I- According to Mobile phase A- Gas chromatography *Gas – solid chromatograph “CSC” *Gas– liquid chromatograph “GLC” B- Liquid chromatography *liquid– liquid chromatography “LLC” *liquid- solid chromatography”LSC” C- Supercritical fluid chromatography * Liquid Chromatography: Separation in liquid chromatography is based on interaction of the sample with the liquid mobile phase & the stationary phase. Compounds to be analyzed are dissolved in an liquid, & most separations take place at room temperature. Thus most drugs, being non-volatile or thermally unstable compounds, can be chromatographed without decomposition. High Performance liquid chromatography (HPLC) is the most widely applied technique in liquid chromatography. 11 * Gas Chromatography (GC): - In gas chromatography the sample is volatilized & transported by the carrier gaseous mobile phase through the stationary phase where separation takes place. - In this respect, residual solvents in drug substances and drug products are suitable for gas chromatographic analysis. - -Suitable for stable volatile analytes 12  Classification of chromatography “II-based on the equipment or stationary phase ” Planar Chromatography Column Chromatography SP: on Planar Surface SP: in narrow tube “Column” “planar surface support” MP: Liquid (LC), Gas(GC) MP: Liquid & forced by & forced through the column by capillary action gravity or pressure 1- Open Tubular Column 1- Paper Chromatography SP: Coated on the inner surface SP: Water adsorbed on filter of the column. paper 2-Thin-Layer Chromatography 2- Packed Column SP: Silica or Alumina coated on SP: Finely Divided particles compressed in column plastic or glass support III- According to mechanism of separation “mech. Of interaction of sample with stationary phase” i- Adsorption Chromatography (sample interact by adsorption on solid stationary phase) ii- Partition Chromatography (stationary and mobile phases are two immiscible liquids and sample interact with solubilization) iii- Ion Exchange Chromatography.... Ion exchange stationary phase that interacts electrostatically used for separation of charged samples 14  iv- Affinity chromatography (interaction between biological sample and stationary phase is specific antigen-antibody reaction) v- Size or steric exclusion (molecular sieving) Chromatography The stationary phase is solid and has controlled pore size and the sample is screened or filtered according to its molecular size, there is no interaction between solute and stationary phase. The large molecules rapidly washed through the column, the smaller molecules penetrate inside the pores and elute later. * Terminolgy - Eluent: Mobile phase..fluid going to stationary phase - Eluate: fluid leaving the stationary phase.. fluid carrying analyte - Elution: movement of mobile phase through stationary phase = Run - Chromatograph: Equipment used for chromatographic analysis & separation - Chromatogram:.Output of a chromatography =Graphical presentation of the separated analytes by chromatography = Detected Pattern of separated analytes formed in a chromatography 16