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CHROMATOGRAPHICAL APPLICATIONS

CHROMATOGRAPHICAL APPLICATIONS. Dr. S. TURE UCL, Imperial College, Ph.D. University of London. Chromatography. Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient of sample components between 2 different phases.

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CHROMATOGRAPHICAL APPLICATIONS

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  1. CHROMATOGRAPHICAL APPLICATIONS Dr. S. TURE UCL, Imperial College, Ph.D. University of London

  2. Chromatography Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

  3. Distribution Coefficient Definition: Different affinity of these 2 components to stationary phase causes the separation. Concentration of component A in stationary phase Concentration of component A in mobile phase

  4. Definition of Chromatography • Simplified Definition: • Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. • Explanation: • Compound is placed on stationary phase • Mobile phase passes through the stationary phase • Mobile phase solubilizes the components • Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

  5. 1 2 Detector Signal time or volume Chromatography Chromatogram - Detector signal vs. retention time or volume

  6. Types of Chromatography Types of Chromatography • Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) • Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) • Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) • Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

  7. Types of Chromatography LIQUID MOBILE PHASE Liquid-Solid Liquid-Liquid (Adsorption) Chromatography FORMAT Chromatography (Partition) Solid Liquid STATIONARY PHASE Reverse Phase Normal Phase Normal Phase Reverse Phase Nonpolar Mobile Phase - Mobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar

  8. Sifat Fisika kimia kertas untuk Kromatografi • Kertas terdiri dari 98 -99 %  selulose, 0,3-10 %  selulose, dan 0,4-0,8 % pentosan. Juga mempunyai gugus karboksilat yang dapat menimbulkan muatan negatif pada kertas. • Kertas kromatografi terdapat kontaminan asam amino yang mempunyai kadar Nitrogen 15 mg/kg kertas.Senyawa lipofilik 25 mg/kg. dan senyawa an organik (kadar abu) 0,04-0,07%, • Senyawa kontaminan tidak mengganggu dalam pemisahan sampel pada kromatografi.Yang penting kemampuan absorbsi dan kenaikkan kapileritas masing-masing kertas. • Whatman no.1 sebagai kertas standard yang digunakan, no. 3MM digunakan untuk preparatif. Sedangkan no. 4 untuk elusi yang cepat, dan 33 ET untuk elusi sangat cepat.

  9. Paper Chromatography Paper chromatography. Molecules separate as they move up the paper. The distance that the molecules travel depends on their size and solubility in the solvent.

  10. Paper Chromatography • Similar to TLC • Stationary phase = H2O adsorbed by cellulose • Mobile phase = solvent • Frequently used to polar compounds • Amino acids, carbohydrates, etc.

  11. Stationary phase: Papers (cellulose), mechanism of separation is through partition. Mobile phase: As TLC but more polar mixtures are usually used. Buffers can also be used. Sample application: A line drawn by pencil, spot places are determined as dots. Apply sample as in TLC.

  12. Paper Chromatography

  13. Types of Paper Chromatography • Radial chromatography • Ascending chromatography • Descending chromatography

  14. Radial Chromatography • In this type of chromatography, as the pigment separates, the different colours move outwards.

  15. Radial Chromatogram

  16. Ascending Chromatography • The solvent moves upwards on the separating media \

  17. Development Type of Ascending: 1- Single development: The solvent system is allowed to move through the stationary phase one time only against gravity. 2- Repeated developments: a- Multiple developments: The plated are developed more than one time using the same solvent system. The plates must be completely dried after each development. b- Stepwise developments: The plated are developed more than one time using different solvent systems.

  18. 3- Two-dimensional development: Is used to verify if a given spot on TLC using the above methods of development (one Dimensional) is one pure compound or mixture of two closely related compounds. The spots are applied to one corner and the plate developed as usual. The plate is then rotated 90 ˚C and then developed again. This method allow better separation of related compounds.

  19. Descending Chromatography • The solvent moves downwards on the separating media.

  20. Pada kromatografi kertas lebih banyak digunakan sistem menurun sehingga lebih cepat perambatan nya. Keuntungan yang lain kromatografi kertas dapat digunakan lembaran kertas yang lebih panjang sehingga dapat dipisahkan campuran yang lebih kompleks. Pemisahan yang terjadi berdasar atas peristiwa partisi, karena fase gerak yang digunakan adalah pelarut organik yang semi polar. Dan umumnya pelarut yang digunakan mengan- dung air sehingga air akan mudah terikat oleh selulosa, dan selulosa dapat mengembang menyerap air, maka air akan berfungsi sebagai fase diam. Komposisi Fase gerak yang dikenal dengan nama BAW (Butanol, Acetic Acid Water). Banyak digunakan untuk pemisahan flavanoid.

  21. Fase gerak yang berupa pelarut organik akan berkompetisi melarutkan sampel yang dianalisis Kromatografi kertas dapat diubah polaritasnya dengan cara inpregnasi atau pembaceman, antara lain dengan asetilasi, foforilasi, fomilasi. Atau dengan senyawa yang bersifat lifofilik seperti parafin, vaselin, undekan. Pembaceman sistemnya seperti pada KLT, hanya pada kromatografi kertas dengan arah yang menurun atau desenden. Dengan cara tersebut kromatografi kertaspun dapat digunakan sebagai kromatografi fase terbalik. Arah elusi dari kertas untuk kromatografi biasa nya ditunjukkan oleh panah, kalau tak ada, digunakan arah yang memanjang dari kertas.

  22. Gambaran fase diam selulose 24

  23. PENGUBAHAN GUGUS HIDROKSIL O-CO- CH3 O OH H H CH2 O-CO- CH3 O-CO- CH3 H HO H O-CO- CH3 • Asetilasi (CH3COOH) • (C6H12O6)n + n x 4 (CH3COOH)  25

  24. PENGUBAHAN GUGUS HIDROKSIL OH OH O OH H H CH2 OH -O-PO4 (OH)2 H HO O-PO4 (OH)2 H • OH • Fosforilisasi (HO-P=O = H3PO4 26

  25. Capillary Action–the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase. Principles of Paper Chromatography

  26. Visualization (Detection of spots): A- Universal methods: 1- Destructive methods: The plated are sprayed with corrosive reagents and then heated in oven where organic compounds will give charred spots. After this treatment the materials can not be recovered. e.g. Anisaldehyde / H2SO4 Vanillin / H2SO4

  27. 2-Non – Destructive methods: In these methods the materials can be recovered. • Day light for colour compounds. • UV light for fluorescent compounds (conjugated double bonds). • I2 vapour for any compounds contain at least one double bond • Spray with water where organic compounds appear as white opaque spots.

  28. B- Specific Methods: • These reagents are used for the detection of certain classes of compounds. They are usually destructive. • Dragendorff΄s reagent for Alkaloids. • Ferric Chloride (FeCl3) for phenolic compounds. • Aniline phthalate for sugars. • Ninhydrine for nitrogenous compounds as Amines, Amino acids.

  29. Tabel Beberapa penggunaan pelacak bercak pada kromatografi kertas Sebyawa fiuoresen Amin ter/kuater. Turunan karbamat HeterosikUk amin kanabinol, sulfonamida Alkaloid/Amin kuar Ter.Heksa(penta klorfenol) Ikatan rangkap, seny.organik 31 31

  30. Rate of flow (RfValue): Distance traveled by the spots Rf= ----------------------------------------- Distance traveled by the solvent The Rf of any compound must be less than one.

  31. Tailing in Paper Chromatography: In some cases instead of getting round spots a Tailed or comet like spots are obtained leading to overlapping of the spots and poor resolution.

  32. Reasons and solution for tailing problem: 1-Ionic characters of acids and bases when they are chromatographed under neutral conditions. Solution: add acids or bases to the developing system. 2-Application of large amounts of material. Solution:decrease conc. of material. 3-Unproper choice of solvent system. Solution:change the solvent system.

  33. Application: 1- Qualitative: • Identification through comparison of the Rfvalue with that of Reference material. • Determination of Complexity of mixtures. That will be indicated from number of spots. • Determination the purity of materials. • Monitoring the progress of Chemical reactions. • Monitoring of column chromatography. • Development of finger print TLC for extracts, volatile oils or pharmaceutical preparation for future identification and comparison. In this application plates 5×5, 5×10 cm with thin film of coating material are usually used.

  34. 2- Quantitative: In this case an accurate volume of samples are applied using syringes. The dimensions of plates range from 5x10 to 20x20 according to the number pf spots used. The plates are developed as usual in the chromatographic tanks. After development the concentration of material can be determined by: • Spot area measurement: Which is directly proportional to the conc. of materials. • Photodensitometry: Measure transmittance, reflection or fluorescence of spots. • Radioactivity: For radioactive material. These measurements are done using TLC Scanner connected to computer that perform all calculations.

  35. Paper Chromatography Experiment What Color is that Sharpie?

  36. Overview of the Experiment Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography. Time Required: Prep. time: 10 minutes Experiment time: 45 minutes

  37. Materials List • 6 beakers or jars • 6 covers or lids • Distilled H2O • Isopropanol • Graduated cylinder • 6 strips of filter paper • Different colors of Sharpie pens • Pencil • Ruler • Scissors • Tape

  38. Preparing the Isopropanol Solutions • Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: • - 0%, 5%, 10%, 20%, 50%, and 100%

  39. Preparing the Chromatography Strips • Cut 6 strips of filter paper • Draw a line 1 cm above the bottom edge of the strip with the pencil • Label each strip with its corresponding solution • Place a spot from each pen on your starting line

  40. Developing the Chromatograms • Place the strips in the beakers • Make sure the solution does not come above your start line • Keep the beakers covered • Let strips develop until the ascending solution front is about 2 cm from the top of the strip • Remove the strips and let them dry

  41. Developing the Chromatograms

  42. Developing the Chromatograms

  43. Observing the Chromatograms 0% 20% 50% 70% 100% Concentration of Isopropanol

  44. Black Dye 1. Dyes separated – purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20% 0% 20% 50% 70% 100% Concentration of Isopropanol

  45. Blue Dye 1. Dye separated – blue 2. Not very soluble in low concentrations of isopropanol 3. Completely soluble in high concentrations of isopropanol 0% 20% 50% 70% 100% Concentration of Isopropanol

  46. Green Dye 1. Dye separated – blue and yellow 2. Blue – Soluble in concentrations of isopropanol >20% 3. Yellow – Soluble in concentrations of isopropanol >0% 0% 20% 50% 70% 100% Concentration of Isopropanol

  47. Red Dye 1. Dyes separated – red and yellow 2. Yellow –soluble in low concentrations of isopropanol and less soluble in high concentrations of isopropanol 3. Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20% 0% 20% 50% 70% 100% Concentration of Isopropanol

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