بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم CHROMATOGRAPHY

2. CONTENTS Introduction Classification of chromatographic methods Principle of chromatography High performance liquid chromatography (HPLC) Gas chromatography (GC) Thin layer chromatography (TLC)

3. Introduction What is chromatography ? Definition: Chromatography is defined as a procedure by which solutes are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density.

4. Mobile Phase: The Phase that travels through the column (gas or liquid) – transport sample through the column. Stationary Phase: Immiscible solid or liquid phase that fixed in place in the column or on a solid support – retain analytes within the column. • Band or Zone: • Area across which analyte is distributed on column • Zones of different analytes gradually separate as bands progress down column

5. Column Chromatography Thin Layer chromatography • Sample • Mobile phase (eluant) • Stationary Phase • Detection method • Chromatogram

6. Liquid Chromatography Gas Chromatography • Method to separate components in a mixture based on different Distribution coefficients between the two phases. • Chromatography categorized on the basis of interaction between solute and stationary phase • Mobile phase either gas or liquid • Stationary phase either liquid or solid • Liq/Liq (Partition) • Liq/Sol (Adsorption) • Gas/Liq (Partition) • Gas/Sol (Adsorption)

7. According to methodology Planer chromatography Column chromatography Thin Layer TLC Paper PC HPLC GC Electrophoresis Classification of chromatographic methods

8. III. PRINCIPLE OF CHROMATOGRAPHY A. Concept of Chromatography: Sample Mobile Figure: Schematic diagram showing the separation of compounds A and B. and the output of the detector response at various stages of elution • The process of: • Addition of sample • Mobile elution process • Separation mechanism • Retention time ? • Detection by, UV lamp, UV detector, other detectors. • Eluted bands / collection • Chromatogram? (function of retention time versus detector response) • Partition coefficient K’ • k’ = Cs/CM A B Response time

9. Thin-Layer Chromatography TLC

10. TLC Chromatography carried out on active particulate material (silica gel or alumina) dispersed on an Inert support (flat glass plates) Principles of (TLC)

11. Preparation of the Plate Sample Application Chromatogram Development Locating of the Spots Basic Steps of TLC Technique

12. Preparation of the Plate • Slurry of the active material is uniformly spread over the plate by means of a commercially available spreader. • Air-drying overnight, or oven-drying at • 80-90 C for about 30 minutes. • Ready to use thin layers (pre-coated plates) • are commercially available.

13. 1-2 cm 1-2 cm Base line 2-2.5 cm Sample Application 

14. Solvent front a b Base line  Locating of the Spots For Colored Compounds: Rf = b/a

15. Where is the spots ?? We do not know. Solvent front a b  Base line   • Iodine or sulphuric acid is used for most organic mixtures. • Ninhydrin is used for amino acids. • 2,4-Dinitrophenylhydrazine is used for aldehydes and ketones For Colorless Compounds: Rf = b/a

16.       Co-spot Co-spot Unknown Unknown Authentic Authentic Applications of TLC Technique Identification of Unknown Compounds

17. Analysis of Reaction Mixture    Product Rxn. mixt. Start. mat.

18.  Chromatogram Development • Avoid direct contact between the sample and the solvent system. • The tank or chamber is preferably lined with filter paper. • As the developing solvent travels up the plate, it dissolves the sample and carries it up; the sample distributing itself between the moving solvent and the stationary phase.

19. Product compound Impurities  Determination of the Purity of a Product Compound

20. Unknown Standard conc. Signal        Concentration Calibration curve Quantitative Determination of an Unknown Concentration

21. Sample injection valve Solvent mixing valve Pump Column HPLC Chart Detector Recorder Mobile phase reservoir Waste Instrumentation of HPLC

22. Type Response Sensitivity (ng/mL) Refractive index Universal 1000 Conductimetric Selective 100 UV/visible absorption Selective 10 Mass-spectrometry Selective 0.1 Fluorescence Selective 0.001 HPLC Detector Characteristics of Typical HPLC Detectors :

23. injection valve Solvent mixing valve Pump Column Chart Recorder Detector Mobile phase reservoir Waste HPLC Recorder

24. Compound Impurity Authentic Unknown What is the Applications of HPLC ? Peaks correspond to individual components Qualitative Analysis Separation of Mixture Components Quantitative Analysis Purification of Compounds Identification of Compounds

25. Calibration curve Peak hight Concentration 0 0 0 0 0 10 0 10 0 10 10 10 10 10 100 g/mL 75 g/mL 50 g/mL 25 g/mL 10 g/mL 5 g/mL Unknown Quantitative Analysis External Standard Method

26. GC Gas Chromatography

27. Instrumentation of GC Flow meter Injector Septum Vent Detector Pressure regulator Flow controller GC Chart Recorder  Oven Gas supply Column

28. GC Column • Packed column • ~ 3-6mm inner diameter tubing, 1-5 m long • used for preparative separations or to separate gases that are poorly retained • lower resolution • small, uniform particle size decreases Eddy diffusion (requiring higher pressures) • open tubular (more common): • 0.1-0.5 mm inner dia., 10-100 m long • 0.1-5 mm thick sp coated on inner walls • higher resolution, shorter analysis times, greater sensitivity compared to packed columns

29. Flame Ionization Detector (FID): cathode (collects CHO+ ions) anode air H2 column effluent Detectors

30. Flame Ionization Detector (FID): organic solutes are burned in flame producing CH radicals and eventually CHO+ CH. + O. CHO+ + e- CHO+ ions are collected by cathode, produces current as the response Detectors

31. Authentic Unknown Applications of GC ? Peaks correspond to individual components Qualitative Analysis Separation of Mixture Components: Quantitative Analysis Identification of Compounds: Retention time comparsion Pyrolysis gas chromatography It is used for the identification of non-volatile materials (plastics, natural and synthetic polymers, and some microbiological materials. It is based on the fingerprint chromatogram for the sample, which results from its thermal dissociation and fragmentation.

32. Calibration curve Peak hight Concentration 0 0 0 0 0 10 0 10 0 10 10 10 10 10 100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown Quantitative Analysis External Standard Method

33. Aspects of GC Applications: Food Analysis Analysis of foods is concerned with confirm the presence and determination the quantities of the analytes (lipids, proteins, carbohydrates, preservatives, flavours, colorants, and also vitamins, steroids, and pesticide residues). Drug Analysis GC is widely applied to identification of the active components, possible impurities as well as the metabolites.

34. Environmental Analysis The environmental contaminants; e.g. dichlorodiphenyltrichloro- ethane (DDT) and the polychlorinated biphenyls (PCBs) are present in the environment at very low concentrations and are found among many of other compounds. GC, with its high sensitivity and high separating power, is mostly used in the analysis of environmental samples. Forensic Analysis In forensic cases, very little sample is available, and the concentration of the sample components may be very low. GC is a useful due to its high sensitivity and separation efficiency.

35. Good Luck mahmoud sheha