Chromatography and its applications by Dr.A.DINESH KARTHIK
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This document provides information about chromatography techniques. It begins with an overview of chromatography and its inventor, Mikhail Tswett. It then describes several common chromatography methods including thin layer chromatography, paper chromatography, gas chromatography, ion exchange chromatography, and discusses their components and principles. Key terms like mobile phase, stationary phase, retention factor (Rf) are explained. Various chromatography applications and advantages are also mentioned.
Chromatography and its applications by Dr.A.DINESH KARTHIK
- 1. Dr.A.DINESH KARTHIK HEAD, P G & RESEARCH DEPT. OF CHEMISTRY SHANMUGA INDUSTRIES ARTS & SCIENCE COLLEGE, TIRUVANNAMALAI-606603.
- 2. Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster. Chromatography
- 3. Invention of Chromatography Mikhail Tswett Russian Botanist (1872-1919) Mikhail Tswett invented chromatography in 1901 during his research on plant pigments. He used the technique to separate various plant pigments such as chlorophylls, xanthophylls and carotenoids.
- 4. Chromatography: (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color. Common Types of Chromatography Tswett’s technique is based on Liquid Chromatography. There are now several common chromatographic methods. These include: Paper Chromatography Thin Layer Chromatography (TLC) Liquid Chromatography (LC) High Pressure Liquid Chromatography (HPLC) Ion Chromatography Gas Chromatography (GC)
- 5. Thin Layer Chromatography Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support. Substances that are less attracted to the solid or are more soluble in the liquid move faster. And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf
- 6. THIN LAYER CHROMATOGRAPHY Stationary Phase ---------> Silica Gel Mobile Phase -------------> Solvent (developing)
- 7. SOLVENT SPOT DEVELOPING CHAMBER Origin Solvent Front 1.1 cm 5.5 cm Rf = Distance from starting origin to center of zone Distance from starting origin to solvent front = 5.5 11 = 0.5 THIN LAYER CHROMATOGRAPHY
- 8. Paper and Thin Layer Chromatography Later The solvent moves up paper by capillary action, carrying mixture components at different rates. solvent solvent front
- 10. Rf = distance moved by substance distance moved by solvent front For substances that are very soluble in the liquid Rf will be close to .... For substances that are rather insoluble in the liquid Rf will be close to .... 1 0
- 11. Comparing Chromatography to the Flow of a River... Base Water flow Light leaf Heavy stone
- 12. How Does Chromatography Work? In all chromatographic separations, the sample is transported in a mobile phase. The mobile phase can be a gas, a liquid, or a supercritical fluid. The mobile phase is then forced through a stationary phase held in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample. The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.
- 13. Mobile Phase / Stationary Phase ● A site in which a moving phase (mobile phase) and a non- moving phase (stationary phase) make contact via an interface that is set up. ● The affinity with the mobile phase and stationary phase varies with the solute. Separation occurs due to differences in the speed of motion. Strong Weak Mobile phase Stationary phase
- 14. Gas Liquid Chromatography Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an involatile liquid held on particles of a solid support.
- 15. Column Chromatography and Planar Chromatography Separation column Packing material Column Chromatography Paper or a substrate coated with particles Paper Chromatography Thin Layer Chromatography (TLC)
- 16. Separation Process and Chromatogram for Column Chromatography Output concentration Time Chromatogram
- 17. Chromatogram tR t0 Intensityofdetectorsignal Time Peak tR : Retention time h A t0 : Non-retention time A : Peak area h : Peak height
- 18. Ion Exchange Chromatography N+ R R R SO3 - + + + ++ ++ + + + Electrostatic interaction (Coulomb force) Anion exchange Cation exchange
- 19. Stationary Phase Used in Ion Exchange Mode ● Base Material – Resin is often used. – Silica gel is also used. ● Cation Exchange Column – Strong cation exchange (SCX) -SO3 - – Week cation exchange (WCX) -COO- ● Anion Exchange Column – Strong anion exchange (SAX) -NR3 + – Week anion exchange (WAX) -NHR2 +
- 20. Dependence of Exchange Capacity of Ion Exchanger on pH of Eluent Exchange capacity Exchange capacity pH 0 7 14 pH 0 7 14 Weakly acidic cation exchanger Strongly acidic cation exchanger Weakly basic anion exchanger Strongly basic anion exchanger Cation exchange mode Anion exchange mode
- 21. Ion Exclusion Chromatography H+ H+ H+ Strong acid ions are repelled by charge and cannot enter the pore. Depending on the level of dissociation, some weak acid ions can enter the pore.
- 22. Relationship between Retention Time and Salt Concentration of Eluent in Ion Exchange Mode Resin ResinResin The exchange groups are in equilibrium with anions in the eluent. An eluent ion is driven away and a solute ion is adsorbed. The solute ion is driven away by an eluent ion and is adsorbed by the next exchange group. If the salt concentration of the eluent increases, the solutes are eluted sooner. Solute ions and eluent ions compete for ion exchange groups.
- 23. Gas Chromatography (GC) *Gas chromatography is a chromatographic technique that can be used to separate volatile organic compounds. *It consists of a flowing mobile phase an injection port a separation column (the stationary phase) an oven a detector.
- 24. In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.
- 25. The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column. Principle
- 26. Mobile phases are generally inert gases such as helium, argon, or nitrogen. The injection port consists of a rubber septum through which a syringe needle is inserted to inject the sample. The injection port is maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture.
- 27. Since the partitioning behavior is dependent on temperature, the separation column is usually contained in a thermostat-controlled oven. Separating components with a wide range of boiling points is accomplished by starting at a low oven temperature and increasing the temperature over time to elute the high-boiling point components. The more volatile (Low Boiling Point / Higher Vapor Pressure) compounds arrive at the end of the column first and pass into the detector
- 28. Components of a Gas Chromatograph Gas Supply: (usually N2 or He) Sample Injector: (syringe / septum) Column: 1/8” or 1/4” x 6-50’ tubing packed with small uniform size, inert support coated with thin film of nonvolatile liquid Detector: TC - thermal conductivity FID - flame ionization detector
- 32. GC Theory To detector Analyte Column + packing Time
- 33. Separations To detector Time 1 Time 2 Time 3
- 35. GC Columns Capillary columnsPacked columns •Typically a glass or stainless steel coil. •1-5 total length and 5 mm inner diameter. • Filled with the st. ph. or a packing coated with the st.ph. •Thin fused-silica. •Typically 10-100 m in length and 250 mm inner diameter. •St. ph. coated on the inner surface. •Provide much higher separation eff. •But more easily overloaded by too much sample.
- 36. 1. Add about 1 tsp of dry/sifted Tide to fill pipet within 1/4” of top 3. Use a plug of fiberfill to hold Tide in place (Tide has been sifted and dried, so keep lid closed on container.) 2. Tap column with a pencil to settle the powder. Filling the Column *Do not compact Tide into column. **Do not leave any dead space at head of column.
- 37. A gas chromatography oven, open to show a capillary column
- 38. GC Detectors After the components of a mixture are separated using gas chromatography, they must be detected as they exit the GC column. Thermal-conduc. (TCD) and flame ionization (FID) detectors are the two most common detectors on commercial GCs. The others are 1. Atomic-emmision detector (AED) 2. Chemiluminescence detector 3. Electron-capture detector (ECD) 4. Flame-photometric detector (FPD) 5. Mass spectrometer (MS) 6. Photoionization detector (PID)
- 39. GC Detectors Cont’d The requirements of a GC detector depend on the separation application. E.g. An analysis may require a detector selective for chlorine containing molecules. Another analysis might require a detector that is non- destructive so that the analyte can be recovered for further spectroscopic analysis. You can not use FID in that case because it destroys the sample totally. TCD on the other hand is non-destructive.
- 40. TCD Detector A TCD detector consists of an electrically-heated wire.The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non- specific and non-destructive.
- 41. ECD Detector Uses a radiactive Beta emitter (electrons) to ionize some of the carrier gas and produces a current between a biased pair of electrodes. When an org. mol. that contains electornegative functional gr., such as halojens, phosphorous and nitro groups, pass by the detector, they capture some of the electrons and reduce the current.
- 42. FID Detector Consists of a hydrogen/air flame and a collector plate. The eff. from the GC column passes through the flame, shich breaks down org. mol. and produces ions. The ions are collected on a biased electrode and produce an elec. sig. Extremely sensitive, large dynamic range.
- 43. MS Detector Uses the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. The general operation of a mass spectrometer is: 1. create gas-phase ions 2. separate the ions in space or time based on their mass to charge ratio 3. Measure the quantity of ions of each mass-to-charge ratio.
- 44. MS Detector Cont’d The ion separation power of an MS is described by the resolution: R = m/Dm Where m is the ion mass and Dm is the difference in mass between two resolvable peaks in a mass spectrum. E.g., an MS with a resolution of 1000 can resolve an ion with a m/e of 100.0 from an ion with an m/e of 100.1.
- 45. GAS LIQUID CHROMATOGRAPHY Principles Partition of molecules between gas (mobile phase) and liquid (stationary phase).
- 46. Gas-liquid chromatography (GLC) Packed columns are fabricated from glass, metal, or Teflon with 1 to 3 m length and 2 to 4 mm in internal diameter. The column is packed with a solid support (100-400 mm particle diameter made from diatomaceous earth) that has been coated with a thin layer (0.1-5 mm) of the stationary liquid phase. Efficiency increases with decreasing particle size as predicted from van Deemter equation. The retention is based on absorption of analyte (partition into the liquid stationary phase) where solutes must have differential solubility in the stationary phase
- 47. Open tubular capillary columns, either WCOT, SCOT are routinely used. In WCOT the capillary is coated with a thin film (0.1- 0.25 mm) of the liquid stationary phase while in SCOT a thin film of solid support material is first affixed to the inner surface of the column then the support is coated with the stationary phase. WCOT columns are most widely used. Capillary columns are typically made from fused silica (FSOT) and are 15 to 100 m long with 0.10 to 0.5 mm i.d.
- 48. The thickness of the stationary phase affects the performance of the column as follows: 1. Increasing thickness of stationary phase allows the separation of larger sample sizes. 2. Increasing thickness of stationary phase reduces efficiency since HS increases. 3. Increasing thickness of stationary phase is better for separation of highly volatile compounds due to increased retention.
- 49. 49 Much more efficient separations can be achieved with capillary columns, as compared to packed columns, due to the following reasons: 1. Very long capillary columns can be used which increases efficiency 2. Thinner stationary phase films can be used with capillary columns 3. No eddy diffusion term (multiple paths effect) is observed in capillary columns
- 50. Gas-solid chromatography (GSC) Gas-solid chromatography is based upon adsorption of gaseous substances on solid surfaces. Distribution coefficients are generally much larger than those for gas-liquid chromatography. Consequently, gas-solid chromatography is useful for the separation of species that are not retained by gas-liquid columns, such as the components of air, hydrogen sulfide, carbon disulfide, nitrogen oxides, and rare gases. Gas-solid chromatography is performed with both packed and open tubular columns.
- 51. Separation and analysis of organic compounds Testing purity of compounds Determine relative amounts of components in mixture Compound identification Isolation of pure compounds (microscale work) Similar to column chromatography, but differs in 3 ways: Partitioning process carried out between Moving Gas Phase and Stationary Liquid Phase Temperature of gas can be controlled Concentration of compound in gas phase is a function of the vapor pressure only. GC also known as Vapor-Phase Chromatography (VPC) and Gas- Liquid Partition Chromatography (GLPC) Gas Chromatography Application
- 52. SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS C C C Detector Response Retention Time 14 16 18 Peak Area (cm ) Sample Concentration (mg/ml) 2 4 6 8 10 0.5 1.0 1.5 2.0 2.5 3.0 2 The content % of C fatty acids = C C + C + C = the content % of C fatty acids14 14
- 53. TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS Response GC Retention Time on Carbowax-20 (min) Mixture of known compounds Hexane Octane Decane 1.6 min = RT Response Unknown compound may be Hexane 1.6 min = RT Retention Time on Carbowax-20 (min)
- 54. GLC ADVANTAGES 1. Very good separation 2. Time (analysis is short) 3. Small sample is needed - ml 4. Good detection system 5. Quantitatively analyzed
- 55. Food and Cancer ● Chemicals that can cause cancer have a wide variety of molecular structures and include hydrocarbons, amines, certain drugs, some metals and even some substances occurring naturally in plants and molds. ● In this way, many nitrosamines have carcinogenic properties and these are produced in a number of ways such as cigarette smoke. ● GC can be used to identify these nitro-compounds in trace quantities.
- 56. Drugs ● There are still numerous GC applications involving both quantitative and qualitative identification of the active components and possible contaminants, adulterants or characteristic features which may indicate the source of the particular sample. ● Forensic analysis frequently users GC to characterize drugs of abuse, in some cases the characteristic chromatographic fingerprint gives an indication of the source of manufacture of the sample or worldwide source of a vegetable material such as cannabis. ● Analytical procedures, chromatographic methods and retention data are published for over 600 drugs, poisons and metabolites. These data are extremely useful for forensic work and in hospital pathology laboratories to assist the identification of drugs.
- 57. DISADVANTAGES OF GAS CHROMATOGRAPHY Material has to be volatilized at 250C without decomposition. R C OH CH3OH H2SO4 O R C O CH3 O CH2 O C R CH O C R CH2 O C R O O O CH3OH O R C O CH3 CH3ONa Fatty Acids Methylester Reflux + 3 Volatile in Gas Chromatography Volatile in Gas Chromatography + +