DSE-2, ANALYTICAL METHODS -Ch-II.pptx
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The document discusses various analytical chromatography techniques. It describes chromatography as separating components through distribution between two immiscible phases, with one stationary and one mobile. The document outlines different types of chromatography including column chromatography, thin layer chromatography, gas chromatography, and ion exchange chromatography. It discusses the principles, techniques, and efficiency of these analytical methods.
DSE-2, ANALYTICAL METHODS -Ch-II.pptx
- 1. Dr. Santanu Chakravorty CHEMISTRY-DSE: ANALYTICAL METHODS IN CHEMISTRY Dr. Santanu Chakravorty Assistant Professor Mathabhanga College 1
- 2. Dr. Santanu Chakravorty Syllabus Chromatography: Classification, principle and efficiency of the technique. Mechanism of separation: adsorption, partition & ion exchange. Development of chromatograms: frontal, elution and displacement methods. Qualitative and quantitative aspects of chromatographic methods of analysis: IC, GLC, GPC, TLC and HPLC 2
- 3. Dr. Santanu Chakravorty Principles of Chromatography Chromatography now refers to any of a diverse group of techniques that effect a separation through a distribution of sample between two immiscible phases. The requirement to distinguish chromatography from other separation technique is that one phase be stationary while the second phase be mobile and percolating through the first phase resulting in selective retention of the components of a mixture by the stationary phase. The stationary phase which may be solid or liquid or may consists of a mixture of a solid and liquid , is finely divided and is fixed in place. The mobile phase, which may be liquid or gaseous, fills the interstices of the stationary phase and is able to flow through the stationary phase. The physical states of the mobile and stationary phases give rise to four basic type of chromatography i. liquid-solid chromatography, (LSC) ii. Liquid-liquid chromatography, (LLC) iii. Gas- liquid chromatography, (GLC) iv. Gas-solid chromatography (GSC) Solid stationary phase also gives rise to low exchange chromatography. Separation of the components, or solutes of a sample results from differences in their rate of adsorption. 3
- 4. Dr. Santanu Chakravorty Classification • The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achieve a satisfactory separation within a suitable time interval. Various chromatography methods have been developed to that end. Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high- pressure liquid chromatography, and affinity chromatography. Types of chromatography • Column chromatography • Ion-exchange chromatography • Gel-permeation (molecular sieve) chromatography • Affinity chromatography • Paper chromatography • Thin-layer chromatography • Gas chromatography • Dye-ligand chromatography • Hydrophobic interaction chromatography • Pseudoaffinity chromatography • High-pressure liquid chromatography (HPLC) 4
- 5. Dr. Santanu Chakravorty Efficiency of Chromatography techniques Chromatographic methods will separate ionic species, inorganic or organic, and molecular species ranging in size from the lightest and smallest, helium and hydrogen, to particulate matter such as single cells . No single configuration will accomplish this, however. Little preknowledge of the constituent of a mixture is required. At its best, chromatography will separate several hundreds of components of unknown identity and unknown concentrations, leaving the components unchanged. Amounts in the parts per billion range can be detected with some detectors. The solute can range from polar to nonpolar—i.e., water-soluble to hydrocarbon-soluble. • Substances of low critical temperature or low molecular weight, such as the gases at laboratory conditions showing dispersive or London intermolecular forces only, are separated with molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to species with high critical temperatures and normal boiling points as high as 400 °C. Substances that are solids at normal laboratory conditions with molecular weights below 1,000 are best separated with liquid-solid or liquid-liquid systems. Lower members of the molecular weight scale range are amenable to supercritical fluid separations. Size- exclusion methods are involved at molecular weights above 1,000. Field-flow fractionation extends the size range to colloids and microscopic particles. • Separations are fast, ranging from analysis times of a few minutes to several hours. The prechromatographic world would have considered a time of several hours to separate multicomponent mixtures to be miraculously fast. Now several hours is considered excessive, and there is much emphasis on increasing speed. 5
- 6. Dr. Santanu Chakravorty Adsorption Chromatography Adsorption chromatography is a type of LC in which chemicals are retained based on their adsorption and desorption at the surface of the support, which also acts as the stationary phase. This method is also sometimes referred to as liquid-solid chromatography. Retention in this method is based on the competition of the analyte with molecules of the mobile phase as both bind to the surface of the support. The degree of a chemical's retention in adsorption chromatography will depend on (1) the binding strength of this chemical to the support, (2) the surface area of the support, (3) the amount of mobile phase displaced from the support by the chemical, and (4) the binding strength of the mobile phase to the support. Electrostatic interactions, hydrogen bonding, dipole-dipole interactions, and dispersive interactions (ie, van der Waals forces) all may affect retention in this type of chromatography. The binding strength of the mobile phase with the support in adsorption chromatography is described by the mobile phase's elutropic strength. A liquid or solution that has a large elutropic strength for a given support will act as a strong mobile phase for that material because this mobile phase will tend to bind tightly to the support and cause the analyte to elute more quickly as it spends more time in the mobile phase. As an example, a relatively polar solvent such as methanol will have higher elutropic strength for a polar support such as silica than a nonpolar solvent such as carbon tetrachloride. In the same manner, a liquid or solution that has a low elutropic strength for a support would represent a weak mobile phase for that support in adsorption chromatography (eg, carbon tetrachloride on silica). Three types of adsorbents are generally used in adsorption chromatography: (1) polar acidic supports, (2) polar basic supports, and (3) nonpolar supports. The most common polar and acidic support used in adsorption chromatography is silica. The surface silanol groups on this support tend to adsorb polar compounds and work particularly well for basic substances. Alumina is the main type of polar and basic adsorbent that is used in adsorption chromatography. Like silica, alumina retains polar compounds, but alumina works especially well for polar acidic substances. Other types of supports that can be used in adsorption chromatography are nonpolar adsorbents such as charcoal and polystyrene. 6
- 7. Dr. Santanu Chakravorty Column Chromatography Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent. Compounds move through the column at different rates, allowing them to be separated in fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross contamination and stationary phase degradation due to recycling column chromatography can be done using gravity to move solvent, or using compressed gas to push the solvent through the column. 7 •In column chromatography the stationary phase is packed into a glass or metal column. •The mixture of analytes is then applied and the mobile phase, commonly referred to as the eluent, is passed through the column either by use of a pumping system or applied gas pressure. •The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or applied as a thin film to the inside wall of the column. •As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.
- 8. Dr. Santanu Chakravorty Column Chromatography Technique • Column chromatography is a technique in which the substances to be separated are introduced onto the top of a column packed with an adsorbent, passed through the column at different rates that depend on the affinity of each substance for the adsorbent and for the solvent or solvent mixture, and are usually collected in solution as they pass from the column at different times. • It is a solid-liquid technique in which the stationary phase is a solid & the mobile phase is a liquid or gas. • It was developed by the American chemist D.T Day in 1900 while M.S. Tswett, the Polish botanist, 1906 used adsorption columns in his investigations of plant pigments. 8
- 9. Dr. Santanu Chakravorty Elution, Eluent and Rf value In a liquid chromatography experiment for example an analyte is generally adsorbed, or bound to an adsorbent in a liquid chromatography column. The adsorbent, a solid phase, is a powder which is coated onto a solid support. Based on an adsorbents composition, it can have varying affinities to hold onto other molecules-forming a thin film on its surface. Elution then is the process of removing analytes from the adsorbent by running a solvents, called an eluent pass the adsorbent/ analyte complex. The eluent is the carrier portion of the mobile phase. It moves the analytes through the chromatograph. In liquid chromatography, the eluent is the liquid solvent, in gas chromatography, it is carrier gas. 9
- 10. Dr. Santanu Chakravorty Elution, Eluent and Rf value Rf is the retardation factor, which is the ratio of the distance traveled by a compound in a mobile phase compared with the distance traveled by the front of the mobile phase itself. It is always greater than or equal to zero, and less than equal to 1. • A chromatogram is first to be developed with a suitable solvent (mobile phase), depending upon the nature of analytes, and the stationary phases. The developed chromatogram is then dried and the positions (migration values) are measured for analytes and the solvent front. The Rf (retardation/retention factor) values can be calculated by using the given procedure using the above experiment. • A prepared sample solution (A+B) is applied on the chromatogram paper and run through a mobile phase. Analyte (A) and (B) separate out because of different affinities with mobile phase (solvent). The relative measurements are taken for the analytes, the solvent front, and the point where the mixture (A+B) was applied. 10
- 11. Dr. Santanu Chakravorty Rf value calculation • For the analyte (A) • Rf = Distance moved by analyte (A) / Distance moved by solvent front Rf = 2.9 / 4.0 = 0.725 • For the analyte (B) • Rf = Distance moved by analyte (B) / Distance moved by solvent front Rf = 1.3 / 4.0 = 0.325 • So, the Rf values for the analytes (A) and (B) are 0.725 and 0.325 11
- 12. Dr. Santanu Chakravorty Thin layer Chromatography Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase and because of differences in solubility in the solvent. By changing the solvents, or perhaps using a mixture, the separation of compounds can be adjusted. Chemists often use TLC to develop a protocol for separation by chromatography and use TLC to determine which fraction contain the desired compounds. Separation of compound is based on the competition of the solute and the mobile phase for binding sites on the stationary phase. For instances, if normal phase silica gel is used as the stationary phase, it can be considered polar. Given two compounds that differ polarity, the more polar compound has a stronger interaction with the silica and is, therefore, better able to displace the mobile phase from the available binding sites. As a consequence, the less polar compound moves higher up the plate. 12
- 13. Dr. Santanu Chakravorty TLC Technique • Thin layer chromatography technique used to separate non-volatile mixtures. This- layer chromatography is performed on a sheet of glass, plastic or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide. This layer of adsorbent is known as the stationary phase. • After the sample has been applied on the plate, a solvent or solvent mixture is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. For example, with silica gel, a very polar substances, non-polar mobile phase such as heptane is used. • After the experiment, the spots are visualized. Often this can be done simply by projecting ultraviolet light onto the sheet; the sheets are treated with a phosphor, and dark spots appear on the sheets were compounds absorb light impinging on a certain area. • To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase. This ratio is called retardation factor. In general, a substance whose structure resembles the stationary phase will have low Rf, while one that has a similar structure to the mobile phase will have high retardation factor. 13
- 14. Dr. Santanu Chakravorty Difference between column chromatography and thin layer chromatography The main differences are: • TLC has a stationary phase of alumina or silica gel. Column Chromatography is packed uses its stationary phase with an appropriate matrix material, such as silica. • TLC is carried for the against gravity. Column Chromatography is run under gravity. • TLC uses for the analytical purpose. Column Chromatography uses for the preparative purpose. • Column Chromatography take more time to separate than TLC. • TLC needs less quantity of solvent to separate the analytes . Column chromatography required more amount of solvent. • TLC needs more polar solvent compared to the column chromatography. 14
- 15. Dr. Santanu Chakravorty Partition Chromatography Partition Chromatography Principle • In partition chromatography, the separation of the components from the sample takes place through the process of partition the components between two phases, where both the phases are present in liquid form. In this procedure, the immiscible solid surface that is covered with the liquid surface on the stationary phase is in the mobile phase. • The stationary phase immobilizes the liquid surface which ultimately changes into a stationary phase. The components are separated just after the mobile phase shifts from the stationary phase. The separation is because of the differences in partition coefficients. 15
- 16. Dr. Santanu Chakravorty Technique • For a simple comprehension of the partition chromatography, here we are explaining the procedure for conducting paper chromatography. Apparatus required: • Chromatography jar, Capillary tube, Stationary phase • Mobile phase – Chloroform, methanol, acetone, or ethanol Steps: • Selecting an appropriate type of development – Based on solvent, mixture, paper complexity, etc. the type of development is selected. Usually ascending paper chromatography is utilized as it is a simple technique to perform and also the chromatograms are quickly acquired. • Choosing a stationary phase – Based on pores size and quality of the analyte, filter paper (stationary phase) is chosen. • Preparing the sample – The sample is prepared by dissolving the component in a proper solvent that is employed during the procedure of producing mobile phases. • Spotting the sample on paper – The sample mixture is applied on the filter paper at an appropriate position. • Developing the chromatogram – With the help of a chromatographic jar, the chromatogram development is determined by drenching the paper into the solvent or mobile phase. Through the capillary action, the mobile phase is allowed to run over the test sample. • Paper drying and identifying the compounds – After the development of the chromatograms, the filter paper is dried with the aid of an air dryer. Paper possessing different bands of molecules can be studied in the UV cabinet. Rf values are also figured out. 16
- 17. Dr. Santanu Chakravorty Ion Exchange Chromatography • IEC is a type of liquid chromatography in which ions are separated by their adsorption onto a support that contains fixed charges on its surface. This method relies on the interaction (or exchange) of ions in the sample or mobile phase with fixed ionic groups of the opposite charge that are bound to the support and act as the stationary phase. Depending on the charge of the groups that make up the stationary phase, the types of ions that bind to the column may be either cations (ie, positively charged ions) or anions (ie, negatively charged ions). These two methods are referred to as cation-exchange chromatography and anion- exchange chromatography, respectively. Supports for cation-exchange chromatography contain negatively charged functional groups. These groups may be the conjugate bases of strong acids, or the conjugate bases of weak acids. The supports used in anion-exchange chromatography are usually the conjugate acids of strongly basic quaternary amines, or the conjugate acids of weak bases. • A strong mobile phase in IEC is usually a mobile phase that contains a high concentration of competing ions. The presence of these competing ions will make it more difficult for a charged analyte to bind to the fixed charges that act as the stationary phase. A weak mobile phase in IEC is one that contains few or no competing ions or that otherwise promotes binding by charged analytes to the column. Changing the competing ion concentration is the most common approach for adjusting the retention of analyte ions in IEC. The retention of ions in this method also may be affected by (1) pH, (2) the type of competing ion used, (3) the type of fixed charges used as the stationary phase, and (4) the density of these fixed charges on the support. 17
- 18. Dr. Santanu Chakravorty Ion Exchange Chromatography Technique Key steps in the ion exchange chromatography procedure are listed below: • An impure protein sample is loaded into the ion exchange chromatography column at a particular pH. • Charged proteins will bind to the oppositely charged functional groups in the resin • A salt gradient is used to elute separated proteins. At low salt concentrations, proteins having few charged groups are eluted and at higher salt concentrations, proteins with several charged groups are eluted. • Unwanted proteins and impurities are removed by washing the column. • A pH gradient can also be applied to elute individual proteins on the basis of their isoelectric point (pI) i.e. the point at which the amino acids in a protein carry neutral charge and hence do not migrate in an electric field. As amino acids are zwitter ionic compounds they contain groups having both positive and negative charges. Based on the pH of the environment, proteins carry a positive, negative, or nil charge. At their isoelectric point, they will not interact with the charged moieties in the column resin and hence are eluted. A decreasing pH gradient can be used to elute proteins using an anion exchange resin and an increasing pH gradient can be used to elute proteins from cation exchange resins. 18
- 19. Dr. Santanu Chakravorty Gas-Liquid Chromatography • GLC is a process by which a mixture is separated into its constituents by a moving gas passing over a liquid stationary phase. Separation of the mixture into its constituents occur by partitioning the sample between a mobile gas phase (known as carrier gas) and a thin layer of non-volatile liquid coated on an inert support. • A gas-liquid chromatograph consists of the following parts: A supply of carrier gas from a high pressure cylinder, sample injection system and flow controller, the column and the detector 19 The sample is introduced as a gas at the head of the column. As a result, components having finite solubility in the stationary liquid phase distributed themselves between this phase and the mobile gas phase, in accordance with
- 20. Dr. Santanu Chakravorty Gas-Liquid Chromatography equilibrium law. Elution is the carried out by forcing an inert gas (eg He, N2, H2 or Ar) through the column. The rate of movement of the various component along the column depends upon their tending to dissolve in the stationary liquid phase. Components having negligible solubility in the stationary phase move more rapidly through the column, while the components whose distribution coefficient favors the solvent liquid phase move slowly through the column. 20 Typical chart record peaks corresponds to individual components detected
- 21. Dr. Santanu Chakravorty Difference between GLC and Column Chromatography Gas chromatography is in principle similar to column chromatography, but has several notable differences. First, the process of separating the compounds in a mixture is carried out between a liquid-stationary phase and a gas mobile phase, whereas in column chromatography the stationary phase is solid and mobile phase is a liquid. Second, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography has no such temperature control. Finally the concentration of a compound in the gas phase is solely function of the vapor pressure of the gas. 21
- 22. Dr. Santanu Chakravorty High Performance liquid Chromatography (HPLC) The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. The specific intermolecular interactions between the molecules of a sample and the packing material define their time “on-column”. Hence, different constituents of a sample are eluted at different times. Thereby, the separation of the sample ingredients is achieved. A detection unit (e.g. UV detector) recognizes the analytes after leaving the column. The signals are converted and recorded by a data management system (computer software) and then shown in a chromatogram. After passing the detector unit, the mobile phase can be subjected to additional detector units, a fraction collection unit or to the waste. In general, a HPLC system contains the following modules: a solvent reservoir, a pump, an injection valve, a column, a detector unit and a data processing unit. The solvent (eluent) is delivered by the pump at high pressure and constant speed through the system. To keep the drift and noise of the detector signal as low as possible, a constant and pulseless flow from the pump is crucial. The analyte (sample) is provided to the eluent by the injection valve. 22