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  1. 1. Chromatography Copyright © 28/05/2013 by Omer Bayazeid. All rights reserved.
  2. 2. Chromatography is a method of physically separating mixtures of gases, liquids, or dissolved substances. Chromatography can be used to identify drugs, poisons and many other substances. Separation is determined by the molecular size and/or charge. Chromatography
  3. 3. General Principles of Chromatography • Separation of molecules by distribution between a stationary phase and a mobile phase. – A stationary phase (absorbent) phase the material on which the separation takes place. can be solid, gel, or liquid. Also called matrix, resin, or beads. – The mobile phase is the solvent transports the sample and it is usually a liquid, but may also be a gas. Also called eluting buffer • The compounds to be separated are considered solutes
  4. 4. The mechanism that causes the stationary phase to retard the movement of molecules: 1. Sieve mechanism  separation according to size or MW. (molecular sieve = gel filtration = size exclusion = gel permeation). 2. Charge interaction  separation based on net charge. Classification by the Separation mode
  5. 5. 3. Solubility characteristics  separation based on polarity Hydrophobic chromatography, reverse-phase chromatography, Adsorption or normal-phase chromatography 4. Biological or Specific interaction  capture any molecule that exhibit such property  affinity chromatography, dye-chromatography  Antibody-antigen: (Immuno precipitations and other forms) Classification by the Separation mode
  6. 6. Classification of Chromatography Gas Chromatography Gas - solid Gas - liquid Liquid chromatography High performance (pressure flow) Thin layer (adsorption) Column (gravity flow) By mobile phase: 1. Liquid chromatography. 2. Gas chromatography.
  7. 7. Type of chromatography Material Paper chromatography Filter paper, cellulose Thin Layer Chromatography Silica gel, alumina, polyamide Gas chromatography Squalene, apezion, carbowax M High Performance Liquid Chromatography C-8, C-18, Licosorb, Silicone
  8. 8. Type of chromatography Solvent Paper chromatography Air, alcohol Thin Layer Chromatography Hexane, ether petroleum, alcohol. Gas chromatography He, Ar, N2 High Performance Liquid Chromatography Cyclohexane, n-hexane, carbon tetrachloride, ethanol, methanol, air
  9. 9. (A) Thin Layer Chromatography:
  10. 10. (A) Thin Layer Chromatography: Is a method for identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material
  11. 11. Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase. A. Thin Layer Chromatography:
  12. 12. A. Thin Layer Chromatography:
  13. 13. If no spots are obvious, the most common visualization technique is to hold the plate under a UV lamp. Many organic compounds can be seen using this technique, and many commercially made plates often contain a substance which aids in the visualization of compounds. Identifying the Spots (visualization)
  14. 14. Alkaloids: Dragendorff’s reagent Cardiac glycosides: Antimony trichloride Sugar: Aniline phthalate Amino acids: Ninhydrin Visualizing Agents
  15. 15. The Rf (retention factor) value for each spot should be calculated. It is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications. Interpreting the Data
  16. 16. Rf values often depend on the temperature and the solvent used in the TLC experiment. The most effective way to identify a compound is to spot known substances next to unknown substances on the same plate. In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots, while a pure sample will show only one spot Interpreting the Data
  17. 17. B. Paper Chromatography
  18. 18. A method of partition chromatography using filter paper strips as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. One is usually water adsorbed on cellulose fibers in the paper. The second is the organic solvent flows past the sample on the paper (stationary phase). B. Paper Chromatography
  19. 19. Definition: A chromatographic analytical separation technique for complex mixtures involving the progressive adsorption of the dissolved component onto a special grade of paper. B. Paper Chromatography
  20. 20. Principle: • The certain solvent are used to separate a mixture ex: water, alcohol. • With capillary action the solvent will move up to filter paper. • Movement of a solvent will bring together component that are separated from the mixture. • Every component that are separated will move to several velocity B. Paper Chromatography
  21. 21. The moving components are depend on : a. Solubility solute in solvent b. Intermolecule forces c. Pore size of filter paper d. Size of solute At the end of process, components that are separated will emerge to different distance on filter paper. Rf values are used to identification of each the component. B. Paper Chromatography
  22. 22. 1. Separation of ink dyes - To compare ink dyes use in any company. 2. Food coloring - To differentiate coloring agent used in food product. 3. Botanist/herbalist - To isolate plant pigment from root and leaves. Use of Paper Chromatography
  23. 23. C. Column Chromatography
  24. 24. This includes chromatographic methods in which: The stationary phase is packed into a column. The mobile phase is a moving liquid. According to the mechanism of separation of solutes, five major types of CC are distinguished. Usually, one mechanism predominates but does not exclude the others C. Column Chromatography
  25. 25. Column Chromatography Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or under the effect of gravity C. Column Chromatography
  26. 26. The analytes interacting most strongly with the stationary phase will take longer to pass through the system than those with weaker interactions. These interactions are usually chemical in nature, but in some cases physical interactions can also be used.
  27. 27. Traditional column chromatography is characterized by addition of mobile phase under atmospheric pressure and the stationary phase is packed in a glass column. Open Column Chromatography (Traditional column chromatography)
  28. 28. Detection: On-column detection for colored or fluorescent compounds directly after developing the chromatogram. Monitoring of eluted fractions (PC or TLC). Using special detectors connected to the column such as refractive index, UV detectors, etc… C. Column Chromatography
  29. 29. High-performance liquid chromatography HPLC
  30. 30. • HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. • Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles. High-performance liquid chromatography HPLC
  31. 31. HPLC System
  32. 32. This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation. Uses Of HPLC
  33. 33. The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column. HPLC Chromatograph injectors
  34. 34. Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible. HPLC columns
  35. 35. The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm. HPLC columns
  36. 36. Column Parameters • Column Material • Deactivation • Stationary Phase • Coating Material Instrument Parameters • Temperature • Flow • Signal • Sample Sensitivity • Detector WHAT AFFECTS SYSTEM
  37. 37. Sample Parameters: • Concentration • Matrix • Solvent Effect • Sample Effect WHAT AFFECTS SYSTEM
  38. 38. • Normal phase . • Reverse phase. • Size exclusion. • Ion exchange. Several column types (can be classified as )
  39. 39. • Absorbance (UV with Filters, UV with Monochromators) • IR Absorbance • Fluorescence • Refractive-Index • Evaporative Light Scattering Detector • Electrochemical • Mass-Spectrometric • Photo-Diode Array Types of Detectors
  40. 40. Gas Liquid Chromatography
  41. 41. Principles: Partition of molecules between gas (mobile phase) and liquid (stationary phase). Gas Liquid Chromatography
  42. 42. Schematic Diagram of Gas Chromatography
  43. 43. 1) Flame Ionization Detector (Nanogram - ng). 2) Thermal Conductivity Detector. 3) Electron Capture Detector. Schematic Diagram of Gas Chromatography DETECTORS
  45. 45. 1.Very good separation. 2.Time (analysis is short). 3.Small sample is needed – ml. 4.Good detection system. 5.Quantitatively analyzed. GLC ADVANTAGES DISADVANTAGES OF GAS CHROMATOGRAPHY • Material has to be volatilized at 250C without decomposition.
  46. 46. • By stationary phase or shape 1.Adsorption. 2.Partition. 3.Ion-Exchange. 4.Molecular Exclusion. 5.Affinity. Classification of Chromatography
  47. 47. Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. 1.Adsorption Chromatography:
  48. 48. This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. 2.Partition Chromatography:
  49. 49. In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. 3.Ion Exchange Chromatography:
  50. 50. Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. 4.Molecular Exclusion Chromatography:
  51. 51. 4.Molecular Exclusion Chromatography:
  52. 52. This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH. 5.Affinity Chromatography:
  53. 53. 5.Affinity Chromatography: