Flash chromatography.pptx

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1. 1 Dr. Pooja Chawla FLASH CHROMATOGRAPHY & ION EXCLUSION CHROMATOGRAPHY

2. 2 FLASH CHROMATOGRAPHY

3. C ONT ENT S Chromatography -- definition -- History -- Types of Chromatography -- Uses of Chromatography Column Chromatography -- Types of Column Chromatography 3

4. CONTENTS Flash Chromatography --definition & history --Column VS Flash Chromatography --Theory --Principle --Selection of Stationary Phase --Adsorbent --Properties of Flash Solvents --Selection of column 4

5. C ONT ENT S Instrumentation of Flash Chromatography --Parts of Flash Chromatography --Pump Systems --Sample Injection System --Columns --Pre Columns --Fraction Collector --Detectors 5

6. C O N T E N T S Advantages Apllications 6

7. W HAT IS C HROM AT OGR A P HY Chromatography is a Greek word chroma “colour” and graphein “to write”. And chromatography is a family of analytical chemistry techniques for the separation of mixtures. It was the Russian botanist “Mikhail Tsvet” who invented the first chromatography technique in 1901. 7

8. T Y P ES OF C HROM AT OGR A P HY Adsorption Chromatography Partition Chromatography Ion Exchange Chromatography Molecular Exclusion Chromatography Affinity Chromatography 8

9. U S ES OF C H ROM AT OGR A P H Y It is used in crime scene investigations In hospitals it can be used to detect alcohol levels in a patient's blood stream It is used for environmental agencies to determine the level of pollutants in water supplies It is used to purify chemicals needed to make a product in a manufacturing plant It is used by pharmacists to determine the amount of each chemical found in each product. 9

10. C OLU M N C HROM AT OGR AP HY In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column. The mobile phase, a liquid, is added to the top and flows down through the column by either gravity or external pressure. Column chromatography is separated into two categories, depending on how the solvent flows down the column. 10

11. W H A T T H E Y ? Depending on how the solvent flows down the column. Gravity column chromatography Flash chromatography If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography . If the solvent is forced down the column by positive air pressure, it is called flash chromatography , 11

12. F L A S H C HROM AT OGR AP HY Flash chromatography, also known as medium pressure chromatography. “ An air pressure driven hybrid of medium and short column chromatography optimized for rapid separation" It was popularized several years ago by Clark Still of Columbia University. An alternative to slow and often inefficient gravity-fed chromatography 12

13. I N T R O D U C T I O N Differs from the conventional technique in 2 ways: Slightly smaller silica gel particles (250-400 mesh) are used, and Due to restricted flow of solvent caused by the small gel particles, pressurized gas ( 10- 15 psi) used to drive the solvent through the column of stationary phase The net result is a rapid “ over in a flash ” and high resolution chromatography. 13

14. COLUMN VS FLASH CHROM T OGRAPHY Cloumn Chromatography Glass columns with silica gel Separation is very slow (typically many hours) End of the run, silica gel must be removed Both time consuming and hazardous Flash Chromatography Pre-packed plastic cartridges Solvent is pumped through the cartridge and separation is rapid Much quicker and more reproducible Remaining solvent flushed out of the column using pressurized gas 14

15. T H E O R Y Chromatography exploits the differences in partitioning behaviour between a mobile phase and a stationary phase to separate the components in a mixture. Compounds of the mixture interact with the stationary phase based on charge, relative solubility or adsorption. The retention is a measure of the speed at which a substance moves in a chromatographic system. 15

16. PRINCIPLE The principle is that the eluent is, under gas pressure (normally nitrogen or compressed air) rapidly pushed through a short glass column. The glass column is packed with an adsorbent of defined particle size with large inner diameter. The most used stationary phase is silica gel 40 –63 μm , but obviously packing with other particle sizes can be used as well. 16

17. PRINCIPLE Particles smaller than 25 μm should only be used with very low viscosity mobile phases, because otherwise the flow rate would be very low. Normally gel beds are about 15 cm high with working pressures of 1.5 – 2.0 bars. In the meantime, however, and parallel to HPLC, reversed phase materials are used more frequently in flash chromatography. The computerized system control the Working of Flash chromatography. 17

18. S E L E C T I O N O F S A T I O N A R Y P H A S E The most important stationary phase in column chromatography is silica. Silica gel ( SiO 2 ) and alumina ( Al 2 O 3 ) are two adsorbents commonly used by the organic chemist for column chromatography. Adsorbent particle size affects how the solvent flows through the column. 18

19. Smaller particles (higher mesh values {70-230} ) are used for flash chromatography; larger particles (lower mesh values { 230- 400 }) are used for gravity chromatography. The amount of silica gel depends on the Rf difference of the compounds to be separated, and on the amount of sample. S E L E C T I O N O F S A T I O N A R Y P H A S E 19

20. A DS OR B ENT S W HIC H A R E M A I N L Y U S E D I N F L A S H C H R O M A T O G R A P H Y Silica : Slightly acidic medium. Best for ordinary compounds, good separation is achieved. Alumina: Basic or neutral medium. Can be effective for easy separations, and purification of amines. Reverse phase silica : The most polar compounds elute fastest, the most non-polars lowest. 20

21. T H E P R O P E R T I E S O F C O MM O N L Y U S E D F L A S H S O L V E N T S : The compound of interest should have a TLC Rf of ≈0.15 to 0.20 in the solvent system you choose. Binary (two component) solvent systems with one solvent having a higher polarity than the other are usually best since they allow for easy adjustment of the average polarity of the eluent. Higher polarity of solvent increases rate of elution for all compounds If your Rf is a≈0.2, you will need a volume of solvent ≈5X the volume of the dry silica gel in order to run your column 21

22. S O LV ENT S Y S T EM S Flash column chromatography is usually carried out with a mixture of two solvents, with a polar and a nonpolar component. 1.Hydrocarbons: pentane, petroleum ether , hexanes. 2. Ether and dichloromethane (very similar polarity) 3. Ethyl acetate 22

23. T H E M O S T C O MM O N T W O - C O M P O N E N T S O L V E N T S Y S T E M Ether/Petroleum Ether Ether/Hexane Ether/Pentane Ethyl Acetate/Hexane Methanol/Dichloromethane 23

24. I N S T R U M E N TA T I O N O F F L A S H C H R O M A T O G R A P H Y 24

25. PA R T S O F F L A S H C H R O M A T O G R A P H Y 1. Pump Systems. -- Pump Controller. 2. Sample Injection Systems. 3.Glass Columns, Filling Sets & Column Valves. 4. Pre columns. 5.Fraction Collector. 6. Detectors and Chart Recorders. 7.Computerize LCD Display. 25

26. P U M P S Y S T E M S Pump Controller :- A pressure range up to either 10 bar or 50 bar gives optimum separation results for a broad range of applications. The pump modules can be controlled by three different units. Pump Controller C610 Pump Manager C615 Control Unit C620 26

27. P U M P C O N T R O LL E R C - 6 1 The Pump Controller C-610 for one Pump Module C-601 is designed for isocratic separations. The flow rate can be easily adjusted by turning a knob and is indicated by a large illuminated LCD-display. Delivered with a overpressure sensor for maximum safety. 27

28. P U M P M A NA G E R C - 615 The Pump Manager C-615 is designed for both isocratic and gradient separations. Fast operation, easy programming and a large graphical display allows a quick and easy set up. The unit has Input/Outputs for 2 solvent valves and level sensors and includes a pressure sensor and mixing chamber. 28

29. C O N T R O L U N I T C - 620 The Control Unit C-620 in combination with Sepacore Control provides precise control of the chromatography system. The following components can be connected to the Control Unit C- 620 2 to 4 Pump Modules C-601 or C- 605 Up to 2 Fraction Collectors Up to 8 Detectors e. g. UV, RIS equential Modules C-623 or C-625 for automatic sequential chromatography on up to 5 columns 29

30. C O L U M N S Glass Columns :- A wide range of columns offer maximum flexibility for every situation. Depending on the nature and the quantity of the sample offers a series of column types which vary in form, size and performance. 30

31. C O L U M N Plastic +Glass Column :- Plastic+Glass-coated Glass Columns are available for larger amounts of samples and higher pressure applications on a high safety level. 31

32. P R E C O L U M N S Pre column are minimizing dead volumes and enhance the life time of the main column by trapping contaminants. The small Pre column, fits to Glass Columns of inner diameter of ID 15, 26, 36 and 49 mm. The large Pre column, fits to Glass Columns of ID 70 and 100 mm inner diameter. 32

33. F I LL I N G S E T S F O R G L A SS C O L U M N S Dry Filling Set :- The Dry Filling Set is employed for filling glass columns with silica gel using compressed gas. Silica gel in the size range of 25 –200micro meters can be packed with this method. 33

34. Slurry Filling Set :- The Slurry Filling Set is used for wet filling and conditioning of glass columns with silica gelparticles smaller than 25 micro meter. 34

35. D E T E C TO R S A N D R E C O R D E R S / S O F T W A R E For most applications one of the robust UV/Vis detectors would be sufficient for the systems detection needs. Both detectors are delivered in combination with a preparative flow cell. UV Monitor C-630 UV Photometer C-635 35

36. A D V A N T A G E S Fast and economic methods for the synthesis laboratory. Ideal for the separation of compounds up to gram quantities. No expensive equipment required. ideal way transfers results from TLC to CLC. Automated changes between normal phase and reversed phase chromatography. 36

37. A P P L I C A T I O N S O F F L A S H C H R O M A T O G R A P H Y: - Natural Products/Nutraceuticals Application: Separation and Isolation of α- Santalol and β- Santalol from Sandalwood Extraction. Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves Extract. Isolation and Purification of Catechins from Green Tea Extract . In Purification of GallaChinensis. Isolation and Purification of Ginsenosides from Red Panax Ginseng Extract. 37

38. Carbohydrate Application Purification of Conjugated Quercetin and Rutinose. Impurity Isolation of Valproic Acid from Cyclodextrine During Encapsulation. Isolation ofAminosugar and Acarbose . Isolation of Aminoglycoside Antibiotics. 38

39. Lipids Application: 1.Purification of Fatty Acid Methyl Esters (FAMEs). 2. Purification of a Mixture of Glycerides, Mono- , Di-, and Tristearin. 3. Purification of Sterols. 39

40. Pharmaceutical/Small Molecules Application In Anti-malarial Drug Purification in Drug Discovery. Mestranol Purification During Chemical Synthesis. In Impurity Isolation During Drug Purification. Bile Acid Purification During Lead Generation in Drug Discovery. 40

41. C O N C L U S I O N Purification of drug is an important step in any branch of research. Preparative chromatography is used to separate the components of a mixture for more advanced use and is thus a form of purification. Flash Chromatography can be alternative to preparative HPLC as it saves time and solvent. 41

42. REFERENCES Yvesrubin.files.wordpress.com. (2019). [online] Available at:https ://yvesrubin.files.wordpress.com/2011/03/flash_chromatography.pdf [Accessed 28 Mar. 2019]. Pharmatutor.org. (2019). [online] Available at: https://www.pharmatutor.org/pdf_download/pdf/Vol.%202,%20Issue%205,%20May%202014,%20PharmaTutor,%20Paper-8.pdf [Accessed 28 Mar. 2019]. Web.mit.edu. (2019). [online] Available at: http://web.mit.edu/5.32/www/Appendix_3_Flash_Cromatography_03.pdf [Accessed 28 Mar. 2019]. Ocw.mit.edu. (2019). [online] Available at: https://ocw.mit.edu/courses/chemistry/5-301-chemistry-laboratory-techniques-january-iap-2012/labs/MIT5_301IAP12_FlashHandout.pdf [Accessed 28 Mar. 2019]. 42

43. ION EXCLUSION CHROMATOGRAPHY 43

44. CONTENTS Ion Exclusion Chromatography Separation mechanism Retention mechanism Optimization Instrumentation Applications 44

45. Ion exclusion chromatography (IEC) is a relatively old separation technique. IEC provides a useful technique for the separation of ionic from non ionic compounds and to separate mixture of acids using an ion exchange stationary phase in which ionic substances are rejected by the resin while non ionic or partially ionized substances are retained and separated by partition between the liquid inside the resin particles and the liquid outside the particles. The ionic substances therefore pass quickly through the column, but non ionic (molecular) or partially ionized substances are held up and are eluted more slowly. IEC is also referred to by several other names, including ion exclusion partition chromatography, ion chromatography-exclusion mode, and Donnan exclusion chromatography. ION EXCLUSION CHROMATOGRAPHY 45

46. The characteristic feature of ion exclusion chromatography is that the sign of the electric charge of the dissociated functional groups on the ion exchange resin is the same as that on the ionic compound analysed . It follows that negatively charged ions, e.g. dissociated acidic compounds, are separated on cation -exchange resins with anionic (usually sulphonic ) functional groups. By analogy, positively charged species are separated on anion- exchange resins containing cationic ( tetra alkyl ammonium functional groups) 46

47. The same column can be used for both ion exchange chromatography and ion exclusion chromatography The eluents used are usually water, water/ organic solvent mixtures, dilute (high conductivity) aqueous solutions of a strong acid, or dilute (low conductivity) aqueous solutions of a weak acid. A conductivity detector is commonly used to monitor the column effluent and, when the eluent conductivity is extremely high, a suitable suppressor system is generally used. Using IEC, it is possible to separate weakly ionized anions such as Sulfide, phosphate, nitrite, aliphatic carboxylic acids, aromatic carboxylic acids, bicarbonate, borate, aliphatic alcohols, sugars, amino acids, water, and others, as well as ammonium, amines, and others, based on a combination of the separation mechanisms of ion-exclusion, adsorption, and/or size exclusion. 47

48. For specific requirement of ion exclusion chromatography, large ion exchange capacity is preferential. Columns capacity is increased by increasing its dimensions, maximizing the concentration of functional groups on the support, and using a strong ion-exchanger. The usual supports are based on macro-porous copolymers of styrene and divinyl benzene in which the degree of cross-linking is characterized by the concentration of divinyl benzene in the reaction mixture. 48

49. In conventional IEC of ionic and non ionic substances, a poly(styrene) divinylbenzene (PS-DVB) based strongly acidic cation exchange resin in the hydrogen form is used exclusively as the separation column. The resin bed can be considered to consist of three distinct components: a solid resin network with charged functional groups (the membrane); occluded liquid with in the resin beads(the stationary phase); and the mobile liquid between the resin beads (the mobile phase or eluent). The ion exchange resin acts as a hypothetical semipermeable membrane (a Donnan membrane) separating the two liquid phases .This membrane is permeable only for non ionic substances. SEPERATION MECHANISM 49

50. When the column is filled with water, which is pumped through as mobile phase, the water soluble molecules build up hydration spheres around the dissociated functional groups of the support. water contained in the pores of the support and in the hydration spheres is immobilized thus forming the stationary phase The basic mechanism is that the neutral and uncharged molecules can penetrate the resin, whereas similarly charged co-ions are repelled owing to the presence of dissociated functional group immobilized in the stationary phase. RETENTION MECHANISM 50

51. Because the concentration of the ions in the stationary phase exceeds that in the mobile phase, osmotic forces tend to drive water into the resin causing it to swell. This swelling is less for more highly cross-linked stationary phases and for mobile phases containing high concentration of ions. The ratio of the concentration of ionized to neutral form of an analyzed compound is determined by its dissociation constant and is equivalent to solute effective charge. Solute retention therefore depends on this constant. Strong acids that are completely dissociated are electronically repulsed . As a consequence they are eluted un separated in the column dead volume ( V M ) , which corresponds to the volume of the mobile phase in the column. 51

52. On the other hand un dissociated molecules are able to enter the resin network. They are eluted together with a retention volume equal to the sum of the inner and dead column volumes. The inner column volume means just the column of the volume of stationary phase. Only electrolytes of the intermediate strength can be separated by this method. For these electrolytes higher retention volumes are expected for species with higher pk a value 52

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54. To optimize an IEC separation, careful selection of the following experimental parameters must be made: The type of matrix used as the stationary phase (PS-DVB, polymethacrylate or silica); The nature of the functional group (e.g. strong or weak acid); The ion exchange capacity (low or high); The nature of the eluent (e.g. strong or weak acids); The pH of the eluent The amount of organic modifier present in the eluent; and The type of detector used (universal or selective). OPTIMIZATION OF ION EXCLUSION CHROMATOGRAPHIC SEPARATION 54

55. INSTRUMENTATION 55

56. Stationary Phases High c a paci t y P S - DV B - b a sed s t r ongly ac i d i c ca t i o n exchange resins of 5 µm particle size. 56

57. Polymethacrylate based weakly acidic cation exchange resin. Unfunctionalized silica gel. Mobile phases: Methanol Ethanol Butanol Glycerol Acetonitrile. 57

58. SUPPRESSOR Reduce the background conductivity of the eluent and enhance the conductivity of the analytes. Minimizes baseline noise Increases the detection sensitivity of the measurement system Optimizes the signal-to-noise ratio 58

59. Dete c tion Conductivity detectors Most popular and universal detection method 59

60. Ions in solution help to transport current. 60

61. Detectors with UV-Visible spectrophotometry Used in cases where the component is absorbed in the UV- Visible range. Eg: iodide, nitrite, nitrate, iodate or chromate ions. Detector is photodiode and the cell is a quartz cuvette. Deute r ium a n d tung s t e n l a m ps are us e d a s a source of light. 61

62. Fluorescence-based detectors Co m p o nen t s o f t h e sa m p l e a r e excited b y a g i v e n wavelength light and the components emit light. Detection of biological samples. 62

63. APPLICATIONS 63

64. V - nitric acid, 1- formic acid 2- acetic acid 3- propionic acid, 4- butyric acid 5- valeric acid 6- caproic acid 7- heptanoic acid 8- caprylic acid 9- pelargonic acid 10- capric acid. SEPARATION OF CARBOXYLIC ACIDS 64

65. Determination of weakly ionized inorganic anions. Fluoride, nitrite, phosphate, sulfite, arsenite, arsenate, bicarbonate, borate and cyanide from seawater and waste waters. Separation of bicarbonate in tap waters by IEC with conductimetric detection by elution with water. (A) Raw tap water (10-fold dilution); tap water after softening treatment; tap water (10-fold dilution). Peaks: 1, strong acid anions; 2, bicarbonate ion. 65

66. Strong Inorganic Acids Sulfate, nitrate and chloride ions, and strong base cations such as sodium, ammonium potassium, magnesium and calcium ions commonly found in acid rainwater. 66

67. Neutral Compounds Neutr a l c o m pounds s uch as suga r s and a lcohols c an be separated by IEC. 67

68. Determination of water in some organic solvents. P S - D VB b a s e d c a t i on e x c han g e re s in i n the h y d r og e n form. Elu e nt - m e t han o l c o n t a in i ng a s m all amou n t of s t r o ng acid. Spectrophotometric detection. Peak at at 310nm. 68

69. Amino acids and amino acid derivatives. were determined using a nd o r otic ac i ds w e r e Hippuric a n d or o t i c acid photodiode array detection. The two pe a ks of hippuric monitored at 210 and 280 nm. 69

70. Głód , Bronisław . (1997). Principles and Applications of Ion Exclusion Chromatography. Acta Chromatographica . 7. 72-87. Pdfs.semanticscholar.org. (2019). [online] Available at: https://pdfs.semanticscholar.org/6e3d/e7af8a62e18f84d8b4b46dad72cc510fdb79.pdf [Accessed 2 Apr. 2019]. REF E RENCES 70

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