Partab chromatography ppt
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It is useful for researchers to evalute the concept of chromatography i m thankful to Dr. Najma Memon associate Prof: NCEAC jamshoro any body can get this ppt by making e-mail at partab_rai2008@yahoo.com
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Partab chromatography ppt
- 1. Liquid Chromatography: University of Sindh Presentation by Partab Rai Research scholar 5 April 2013
- 2. Outline Introduction Principle Variables that affect column efficiency Existing approaches to improve LC efficiency Conclusions References
- 3. Introduction
- 4. What is Chromatography? Background March 8th, 1903, Tswett emphasizes: “The method is based on the property of dissolved substances to produce physical adsorptive compounds with various mineral and organic solid substances”. 1906, Tswett names the newly discovered method: “plant pigments separated on chalk columns (like light spectrum). He called such a preparation as chromatogram, and the corresponding method as chromatographic method”. Michael Semenovich Tswett 1993, IUPAC formulates: “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction”.
- 6. The separation of a mixture by distribution of its components between a mobile and stationary phase over time. Preparative - purify and collect one or more components of a sample Analytical - determine chemical composition of a sample
- 7. Why use Chromatography? Separation of chemical components. Specificity to a particular compound. Purification of chemical components. Identification of chemical components. Quantification of chemical components. Great versatility (a variety of separation modes).
- 8. Chromatography today More than sixty variants of the technique have been developed. HPLC, GC, SFC, and CE are the most frequently used. GC is preferred to HPLC for analysis of gases, however approximately 75% of all known compounds cannot be separated by GC. SFC is higher efficiency than HPLC, but it is limited to non- polar molecules when carbon dioxide is used. Capillary Electrophoresis (CE) is a rival to HPLC, nevertheless the detection sensitivity is much lower than in HPLC.
- 9. Separation is based on the analyte’s relative solubility between two liquid phases Mobile Phase Stationary Phase Solvent Bonded Phase Partitioning :-
- 10. ANALOGY…
- 11. Chromatography Techniques Chromatography Partition Chromatography Adsorption Chromatography • components distribute between 2 immisible liquid phase • components adsorb on solid • relative solubility in 2 phase stationary phase • bonds strongly to mobile phase – move faster Stationary Liquid phase Y X O- Stationary phase Y Stationary phase Mobile Y O- • solid X X Y has a layer of liquid liquid phase Y O- • AI2O3 Mobile X X containing O- • SiO2 liquid phase X and Y O- Y containing X X and Y Y
- 12. Normal Phase. - Polar statioNary Phase aNd NoN-Polar solveNt. reverse Phase. - NoN-Polar statioNary Phase aNd a Polar solveNt.
- 14. A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample travel through the column more slowly than others resulting in the desired separation. Separation is carried by elution process
- 15. InstrumentatIon :- Gradient Controller • Pump Column Detector Injector
- 16. The process of extracting a substance that is adsorbed to another by washing it with a solvent.
- 17. Elution in Column Chromatography Stationary Mobile phase phase t0 t1 t2 t3 t4
- 18. Intermolecular Interactions δ+ δ- δ+ δ- δ+ δ- δ- δ+ a) Dispersion b) Polar (induced dipole-induced dipole) (dipole-dipole, dipole-induced dipole) NO2 NO2 H c) Hydrogen bonding d) Charge transfer e) Ionic
- 19. 1) Solvent reservoirs, 2) Solvent degasser, 3) Gradient valve, 4) Mixing vessel for delivery of the mobile phase, 5) High-pressure pump, 6) Switching valve in "inject position" Switching valve in "load position", 7) Sample injection loop, 8) Pre-column ( guard column), 9) Analytical column, 10) Detector (i.e. IR, UV), 11) Data acquisition, 12) Waste or fraction collector.
- 20. LIquId ChromatographIC CoLumn Smooth-bore stainless steel or heavy-walled glass tubing Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500) Add columns together to increase length
- 21. For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 µL Sampling loops interchangeable loops (5-500 µL at pressures up to 7000 psi)
- 22. Mostly optical Equipped with a flow cell Focus light beam at the center for maximum energy transmission Cell ensures that the separated bands do not widen
- 23. Chromatogram Column Column Chromatogram Time
- 24. Solvents & adsorbents Since adsorbents are polar, non-polar elute first. Usually, the elusion order is as: alkyl halids < saturated hydrocarbons< unsaturated hydrocarbons <ethers < esters < ketones < amines < alcohols < phenols < acids and bases. Polymeric compounds and salts often don’t elute The solvents in the order of polarity Hexane/Pet ether < CCl 4 < toluene < dichloromethane < chloroform < diethyl ether < acetone < ethyl acetate < propanol < ethanol < methanol < acetic acid < water
- 26. Conclusions
- 27. L C used for samples: containing large molecules/ionic containing substances with low vapor pressure (non-volatile substances) Substances thermally unstable Substances can’t be vaporized without decomposing
- 28. Thank You! Waters Acquity BEH (UPLC) Agilent HPLC-Chip Tswett’s column column
Editor's Notes
- Separations of complex mixtures on glass columns (tubes) packed with solid adsorbents, using liquids as eluents were introduced by a Russian botanist Michael Semenovich Tswett when he was carrying out experiments on chlorophyll extracts. His invented technique not only opened the door to understanding the mystery of the green leaf, but served as the basis to a new separation technique – chromatography. In his report “On a new category of adsorption phenomena” that was presented at the meeting of the Biological department of Warsaw Society of Natural Scientists on March 8 th , 1903, Tswett emphasized the development of a new separation method of substances dissolved in organic solvents: In his next paper published 3 years later, Tswett gives a name to this method and calls it chromatography: The term chromatography is coming from the Greek chroma (color) and graphein (to write). However, Tswett does not provide the origin of his term in his publications, and since tswett means color in Russian, it is possible that he named the method after himself, literally Tswett’s writing. Chromatography has evolved from Tswett’s time and now it’s comprised from a wide range of techniques that are based on Tswett’s simple separation process. Unified definition and nomenclature of chromatography was published in 1993 by IUPAC
- Chromatography is one of the most eminent discoveries of the 20 th century, and at the present it is one of the most important analytical techniques. Discoveries in biochemistry, biotechnology, medicine, agriculture, as well as in space science and industry would not have been possible without separation of complex mixtures. Separation of chemical components is essential in any type of chemical analysis as it allows purification as well as qualitative and quantitative measurements. Nowadays chromatographic separations possess such essential valuable characteristics as well-understood separation mechanisms, ease of use, sensitivity, selectivity, robustness, and they’re relatively fast. No other separation method is as powerful as a chromatographic method is, and only a few chemical analysis methods can be found specific to a particular compound.
- Chromatography significantly evolved since its discovery, and now more than sixty variants of the technique have been developed. Gas Chromatography ( GC ) is preferred to HPLC for analysis of gases, thermally stable low-boiling and higher boiling compounds due to GC being more efficient than HPLC. However, GC is applicable to samples volatile below 300ºC, and thus is not applicable to nonvolatile or very-high-boiling compounds. That is approximately 75% of all known compounds can’t be separated by GC. Supercritical Fluid Chromatography ( SFC ) utilizes columns and equipment similar to HPLC, however, the mobile phase is a supercritical fluid, usually a gas under critical pressure and temperature. In terms of separation efficiency, SFC takes place between HPLC and GC. Significant advantages of SFC over HPLC is that SFC can be used in separation of polymeric mixtures, while HPLC is unable to resolve polymeric species with high molecular weights, and that SFC is faster than HPLC since lower viscosity of the mobile phase allows operation at higher flow rates. Capillary Electrophoresis ( CE ) not being a form of chromatography, this separation technique is a rival to HPLC. The separation principle is based on the separation of charged compounds in the order of their mass-to-charge ratios (m/z) in a capillary, under the influence of an electric field. CE, as well as GC and SFC, is characterized by higher than HPLC separation efficiency. The drawback of CE is that its detection sensitivity is much lower than in HPLC. Regardless of HPLC being less efficient than some of the separation techniques, it is still a dominating technique routinely used in the industry due to its essential valuable characteristics comprised of well-understood separation mechanisms, ease of use, sensitivity, selectivity, precision and robustness. This technique has many applications including separation, identification, purification, and quantification of numerous biological and pharmaceutical mixtures of ever increasing complexity. Therefore, in the past decade, significant efforts have been undertaken to improve the efficiency of high-performance liquid chromatography, and successful results were achieved with the use of high temperature LC, monolithic and fused-core columns, and with the use of small-diameter (sub - 2 µm) stationary phase particles. Chromatographic process, the factors influencing chromatographic efficiency in HPLC and the means to improve efficiency are reviewed herein.
- Separation of the components of a mixture takes place in a narrow-bore tubing (column) packed with fine inert solid particles that hold stationary phase. The mobile phase surrounds particles as it percolates through the column. Sample mixture applied to the column as a discrete band (to in Figure 2) carried through the column by the flowing mobile phase. Sample components distribute themselves between the mobile phase and the stationary phase. Elution is promoted by continuous addition of fresh mobile phase into the column. The eluent moves down the column where further partitioning between the stationary and mobile phases occur (t1 in Figure 2) and a new equilibrium establishes. Components in the mobile phase adsorb onto the stationary phase, and components from the stationary phase migrate into the mobile phase (t2 in Figure 2). This process is repeated many times during the elution that eventually leads to separation (t3 in Figure 2) and detection (t4 in Figure 2) of components. Separation of components arises from differential retention of the solutes by the stationary phase. Components that prefer to reside in the stationary phase move down the column slower than those that prefer the mobile phase since solute movement can occur only in the mobile phase. This phase preference can be expressed by the distribution coefficient, K. The net retention of a solute is determined by molecular interactions solute-solute, solute-stationary phase, solute-mobile phase, and stationary-mobile phase interactions. Molecular interactions result from intermolecular forces which are all electrical. Gravitational and magnetic forces may be present, however, they are significantly weaker and therefore don’t affect solute retention. There are three types of intermolecular forces: 1) dispersion , 2) polar , and 3) ionic . Understanding of molecular interactions should allow prediction of retention depending on molecular structure.
- Dispersion forces, also called induced dipole-induced dipole forces or Van der Waals forces, result from charge fluctuations that originate from electron-nuclei vibrations. Iduced dipole induces a dipole in the adjacent atom. The original transient dipole favor electrostatic attraction with the induced dipole. Polar forces occur in a polar molecule that contains a dipole in the form of localized charges located on different parts of the molecule.These charges interact with opposite charges on other molecules. These interactions are always accompanied by the dispersive interactions. Polar forces include dipole-dipole (or orientation), dipole-induced (or induction) interactions, hydrogen bonding and charge transfer interaction. Hydrogen forces are specific to molecules containing oxygen, nitrogen or fluorine atom attached to hydrogen. Hydrogen Bonding arises from the attraction between the slightly positive charge on a hydrogen atom and can be described as a strong dipole-dipole attraction between an electronegative atom and a hydrogen atom bonded to another electronegative atom. Charge transfer or interactions result from the partial transfer of an electron between electron-rich and electron-poor molecules. Such interactions occur between aromatic or unsaturated compounds. Ionic interactions occur between charged ions (counter-ions). Ionic interactions are always accompanied by the dispersive interactions, and sometimes by polar interactions
- As sample components progress down the column, its molecules tend to spread out and occupy larger volumes within the column. The volume that is occupied by a compound’s molecules is called a band . When such band leaves the column (elutes), it is recorded by a detector as a peak in the chromatogram.