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Qualitative and Quantitative Analysis qualitative and quantitative analysis Russian scientist Tswett in 1906 used a glass columns packed with divided CaCO3(calcium carbonate) to separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one. Tswett was the first to use the term "chromatography" derived from two Greek words "Chroma" meaning color and "graphein" meaning to write. Invention of Chromatography by M. Tswett Ether Chromatography Colors Chlorophyll CaCO3 5 *Definition of chromatography *Tswett (1906) stated that „chromatography is a method in which the components of a mixture are separated on adsorbent column in a flowing system”. *IUPAC definition (International Union of pure and applied Chemistry) (1993): Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction. *Principles of Chromatography * Any chromatography system is composed of three components : * Stationary phase * Mobile phase * Mixture to be separated The separation process occurs because the components of mixture have different affinities for the two phases and thus move through the system at different rates. A component with a high affinity for the mobile phase moves quickly through the chromatographic system, whereas one with high affinity for the solid phase moves more slowly. *Forces Responsible for Separation * The affinity differences of the components for the stationary or the mobile phases can be due to several different chemical or physical properties including: * Ionization state * Polarity and polarizability * Hydrogen bonding / van der Waals’ forces * Hydrophobicity * Hydrophilicity * The rate at which a sample moves is determined by how much time it spends in the mobile phase. *Classification of chromatographic methods Chromatographic methods are classified according to: A – Mechanism of separation: The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into: * 1- Adsorption Chromatography: It is the oldest technique. Separation is due to difference in the adsorption power of mixture components. The stationary phase is a solid with adsorption characters. Silica gel and alumina are the most common stationary phase in adsorption chromatography. * 2- Partition Chromatography: Separation is due to difference in solubility of components in two immiscible liquids. The stationary phase is a liquid thin film on an inert solid support. The stationary liquid is usually more polar than the mobile phase. Cellulose powder and wetted silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. * 3- Ion Exchange Chromatography (IEC): It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. * 4- Molecular Exclusion ( Size Exclusion) Chromatography: Separation is based on molecular size. Stationary phase is a material of controlled pore size. Molecules that are very small in relation to the pore size all behave similarly and these small molecules are also not separated. Medium sized molecules are separated based on how far they penetrate into the gel beads. * 5- Affinity Chromatography: The separation is based on the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose - agarose – dextran. B- According to the nature of the mobile and stationary phase: In this regard chromatography is classified into: 1- Liquid Chromatography (LC): The mobile phase is liquid. 2- Gas Chromatography (GC) The mobile phase is an inert gas nitrogen or helium. C- According to the technique (methods of holding the Stationary Phase): 1- Planar or Plane Chromatography: *In this type the stationary phase is used in the form of layer. Plane chromatography is additionally classified into: a- Thin Layer Chromatography (TLC): The stationary phase is spread on glass or plastic or aluminum sheets. b- Paper Chromatography (PC): A specific type of papers is used as stationary phase. 2- Columnar or Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal (gel – ion exchange – adsorption). D- ACCORDING TO PURPOSE OF USE: QUALITITATIVE CHROMATOGRAPHY In this case Chromatography can be used to: 1- Confirm the absence or probable presence of certain constituent in the sample under investigation 2- Give an idea about the complexity of the mixture and the least number of compounds present. 3- Check purity and identity of any compound. QUANTITATIVE CHROMATOGRAPHY The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications. quantitative and qualitative liqiud chromatography *Thin layer chromatography *qualitative analysis After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes moved the TLC plate at different rates, separation is achieved. 2.0 cm R (A) = = 0.40 f 5.0 cm Solvent Front R (B) = 3.0 cm = 0.60 f 5.0 cm Distance solvent migrated = 5.0 cm 4.0 cm Distance A 0.8 cm R (C) = = 0.16 migrated = 3.0 cm f 5.0 cm Distance B migrated = 2.0 cm 4.0 cm 3.0 cm R (D) = = 0.80 f 5.0 cm Distance C migrated = 0.8 cm 0.8 cm R (U ) = 3.0 cm = 0.60 f 1 5.0 cm Origen x x x x x A B U C D 0.8 cm R (U ) = = 0.16 f 2 5.0 cm The Rf retardation factor is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin) The three substances have the same Rf 1 2 3 sample standards The three substances have the same colour 1 2 3 sample The three substances are…invisible 1 2 3 sample *Visualization Method *Most of the time, the spots won’t show unless they are visualized! *Visualization is a method that is used to render the TLC spots visible. *A visualization method can be: *Ultraviolet light *Colored reagents to stain spots *ULTRAVIOLET LAMP After developing a TLC plate, the first analysis technique should always be UV light. This technique is fast. When using "F254 silica gel" TLC plates (silica gel that fluoresces with a 254 nm absorption) these compounds will appear as dark spots (because they "block" the fluorescence by absorbing the UV light) on a green background. Colored reagents to stain spots A TLC stain is used in TLC development to reveal compounds that are not visible by UV. Also, selective detection of compounds is possible by choosing the appropriate TLC stain. *TLC SCANER Classical densitometry uses monochromatic light and a slit of selectable length and width to scan the tracks of a chromatogram, measuring the diffusely reflected light. The CAMAG TLC Scanner uses the entire spectral range from 190 to 900 nm with high spectral selectivity for data acquisition. Absorption spectra for substance identification and for selection of the most suitable measurement wavelength can be recorded within this range. Thin-Layer Chromatography: Qualitative Analysis 1 2 3 sample Densytometr can automatically record spectra as soon as all peak positions are known. The difference beetwen spectra allows us to confirm the identity of analytes in a sample. However, some spectra have small differences and cannot be absolute confirmations by themselves. Analytical chemists use both retention time and spectra to determine a probability of identifying a chemical in a sample. Caffeine spectrum Paracetamol spectrum The Mass Spectrometer In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are: 1. A small sample is ionized, usually to cations by loss of an electron. The Ion Source 2. The ions are sorted and separated according to their mass and charge. The Mass Analyzer 3. The separated ions are then measured, and the results displayed on a chart. The Detector Sample + _ Ionizer Mass Analyzer Detector Sample + _ *Mass Spec Principles Ionizer Mass Analyzer Detector The versatile instrument to extract compounds from a TLC/HPTLC plate and feed them into a mass spectrometer for substance identification or structure elucidation. http://www.google.pl/url?sa=i&source=imag es&cd=&cad=rja&docid=wGxtCaD2i- aHdM&tbnid=lYU4DocqZYlOKM:&ved=0C AgQjRwwADhI&url=http%3A%2F%2Fwww .sciencedirect.com%2Fscience%2Farticle %2Fpii%2FS0021967312013398&ei=ws2b Ub3HBoTCtAb_nYDIBg&psig=AFQjCNFT YgLztE_wmKaWEkYYS5v6Aj3u_Q&ust=1 369251650161134 quantitative analysis Classical densitometry uses monochromatic light and a slit of selectable length and width to scan the tracks of a chromatogram, measuring the diffusely reflected light. The CAMAG TLC Scanner uses the entire spectral range from 190 to 900 nm with high spectral selectivity for data acquisition. Absorption spectra for substance identification and for selection of the most suitable measurement wavelength can be recorded within this range. concentration ug/L 0,6 0,9 1,2 1,5 AU 20 30 40 50 60 50 40 AU 30 20 10 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 concentration ug/L HPLC *What does HPLC means? High Performance Liquid Chromatography High Pressure Liquid Chromatography High Price Liquid Chromatography High Patience Liquid Chromatography Smaller column particle size can improve chromatographic resolution, but increased solvent delivery pressure is needed.
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