Separation Techniques PDF

Separation techniques BCMC 2304 Prof. Dr. Faten A. Nour El-Dien Introduction About the class  Overall aims of course Topics of CA 623 Calendar Prof. Faten A. Nour El-Dien 11/3/2024 BCMC 2304 : Separation Techniques Separation Techniques: (Brief about the meth...

Separation techniques BCMC 2304 Prof. Dr. Faten A. Nour El-Dien Introduction About the class  Overall aims of course Topics of CA 623 Calendar Prof. Faten A. Nour El-Dien 11/3/2024 BCMC 2304 : Separation Techniques Separation Techniques: (Brief about the methods and applications), Chromatographic process, High Performance Liquid Chromatography , Gas Chromatography, Supercritical Fluid Chromatography (SFC), Electrophoresis. Prerequisite: BCMC 1102  References “Fundamentals of Analytical Chemistry” Skoog. “Analytical Chemistry – An Introduction” Skoog, West, Holler, Crouch. “Analytical Chemistry” Gary D. Christian “ Modern Analytical Chemistry” David Harvey Prof. Faten A. Nour El-Dien 11/3/2024 Introduction to Analytical Separations Separations are extremely important in synthesis, in industrial chemistry, in the biomedical sciences, and in chemical analyses. The goals of an analytical separation are usually to eliminate or reduce interferences so that quantitative analytical information can be obtained from complex mixtures. 11/3/2024 Prof. Faten A. Nour El-Dien An interferent is a chemical species that causes a systematic error in an analysis by enhancing or attenuating the analytical signal or the background.  Separations can be complete or partial. Prof. Faten A. Nour El-Dien 11/3/2024 The basic principles of a separation are depicted in Figure. As shown, separations can be complete or partial. In the separation process, material is transported while its components are spatially redistributed. Separations can also allow identification of the separated constituents if appropriate correlations are made or a structurally sensitive measurement technique is used. Prof. Faten A. Nour El-Dien 11/3/2024 The table lists several separation methods that are in common use including (1) chemical or electrolytic precipitation, (2) distillation, (3) solvent extraction, (4) ion exchange, (5) chromatography, (6) electrophoresis, and (7) field-flow fractionation. Prof. Faten A. Nour El-Dien 11/3/2024 Mechanical phase separation An analyte and an interferent can be separated if there is a significant difference in at least one of their chemical or physical properties. Prof. Faten A. Nour El-Dien  Separations Based on Size 1- Filtration: Separations by precipitation require large solubility differences between the analyte and potential interferents. Filtration: Particulate interferents can be separated from dissolved analytes by filtration, using a filter whose pore size retains the interferent. Prof. Faten A. Nour El-Dien 11/3/2024 2- Dialysis: Another example of a separation technique based on size is dialysis, in which a semipermeable membrane is used to separate the analyte and interferent. Illustration of a dialysis membrane in action. In (a) the sample solution is placed in the dialysis tube and submerged in the solvent. (b) Smaller particles pass through the membrane, but larger particles remain within the 11/3/2024 dialysis tube. Prof. Faten A. Nour El-Dien Dialysis membranes are usually constructed from cellulose, with pore sizes of 1–5 nm. The sample is placed inside a bag or tube constructed from the membrane. The dialysis membrane and sample are then placed in a container filled with a solution whose composition differs from that of the sample. Prof. Faten A. Nour El-Dien 11/3/2024  If the concentration of a particular species is not the same on the two sides of the membrane, the resulting concentration gradient provides a driving force for its diffusion across the membrane. Although small particles may freely pass through the membrane, larger particles are unable to pass. Dialysis is frequently used to purify proteins, hormones, and enzymes. During kidney dialysis, metabolic waste products, such as urea, uric acid, and creatinine, are removed from blood by passing it over a dialysis membrane. Prof. Faten A. Nour El-Dien 11/3/2024 3- Size exclusion : is a third example of a separation technique that uses size as a means for effecting a separation. In this technique a column is packed with small porous polymer beads of cross-linked dextrin or polyacrylamide. The sample is placed into a stream of solvent that is pumped through the column at a fixed flow rate. Prof. Faten A. Nour El-Dien 11/3/2024  Size-exclusion Chromatography Prof. Faten A. Nour El-Dien 11/3/2024 Those species too large to enter the pores pass through the column at the same rate as the solvent. Species entering into the pores take longer to pass through the column, with smaller species needing more time to pass through the column. Size-exclusion chromatography is widely used in the analysis of polymers, and in biochemistry, where it is used for the separation of proteins. Prof. Faten A. Nour El-Dien 11/3/2024 Separations Based on Mass or Density If the analyte and the interferent differ in mass or density, then a separation using centrifugation may be possible. The sample is placed in a centrifuge tube and spun at a high angular velocity, measured in revolutions per minute (rpm). The sample’s constituents experience a centrifugal force that pulls them toward the bottom of the centrifuge tube. Prof. Faten A. Nour El-Dien 11/3/2024 The species experiencing the greatest centrifugal force has the fastest sedimentation rate, and is the first to reach the bottom of the centrifuge tube. If two species have equal density, their separation is based on mass, with the heavier species having the greater sedimentation rate. If the species are of equal mass, then the species with the largest density has the greatest sedimentation rate. Prof. Faten A. Nour El-Dien 11/3/2024  Separations Based on a Change of State Because an analyte and its interferent are usually in the same phase, we can achieve a separation if one of them undergoes a change in its physical state or its chemical state. When the analyte and the interferent are miscible liquids, a distillation may be possible if their boiling points are significantly different. 11/3/2024 Prof. Faten A. Nour El-Dien  Typical experimental set-up for (a) a simple distillation, and (b) a fractional distillation. A more efficient separation is achieved by a fractional distillation. Prof. Faten A. Nour El-Dien 11/3/2024 Separations Based on a Partitioning Between Phases The most important group of separation techniques uses a selective partitioning of the analyte or interferent between two immiscible phases. If we bring a phase containing a solute, S, into contact with a second phase, the solute partitions itself between the two phases, as shown by the following equilibrium reaction. The equilibrium constant for reaction is called the distribution constant or partition coefficient. Prof. Faten A. Nour El-Dien 11/3/2024 Liquid–Liquid Extractions A liquid–liquid extraction is usually accomplished with a separatory funnel. After placing the two liquids in the separatory funnel, we shake the funnel to increase the surface area between the phases. Prof. Faten A. Nour El-Dien 11/3/2024 The improved efficiency of multiple extractions falls off rapidly as a total fixed volume is subdivided into smaller and smaller portions. We see that there is little to be gained by dividing the extracting solvent into more than five or six portions. Prof. Faten A. Nour El-Dien 11/3/2024  Ion Exchange Based upon exchange equilibria between ions in solution and ions of like sign on the surface of an insoluble , high-molecular-weight solid. Clay and zeolites , synthetic ion-exchange resins. 11/3/2024 Prof. Faten A. Nour El-Dien Structure of a cross linked polystyrene ion-exchange resin Cation-exchange resins are the sulfonic acid groups-SO3-H+ , a strong acid the carboxylic acid group –COO-H+ , a weak acid.  xRSO3-H+ + Mx+ = (RSO3-)x Mx+ + xH+ solid solution solid solution Anionic exchange contain tetiary amine groups –N(CH3)+3OH- , strong base primary amine groups –NH+3OH- ; a weak one. xRN (CH3)3OH- + Ax- = [RN (CH3)3+ ]x Ax- + xOH- solid solution solid solution Prof. Faten A. Nour Eldien  Chromatography Chromatography is a widely used method for separation, identification and determination of chemical components in complex mixtures.  Drugs  Chocolate Chemicals  Pharmaceutical Purity  5% of all chemical research uses chromatography  50% of industrial chemistry uses chromatography Prof. Faten A. Nour El-Dien 11/3/2024   Early 1900s botanist Mikhail Tswett separating leaf pigments.  He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of Pioneering work by Martin plant pigments such as: http://upload.wikimedia.org/wikipedia/commons/thumb/d/d4/Chromatography_of_chlorophyll_-_Step_7.jpg/75px-Chromatography_of_chlorophyll_-_Step_7.jpg and Synge in 1941 established the importance of  Chlorophyll liquid–liquid partition  Carotenes chromatography  Xanthophylls Sample separated into zones or bands 11/3/2024 Prof. Faten A. Nour El-Dien  The history of modern chromatography can be traced to the turn of the century when the Russian botanist Mikhail Tswett (1872–1919) used a column packed with a stationary phase of calcium carbonate to separate colored pigments from plant extracts. The sample was placed at the top of the column and carried through the stationary phase using a mobile phase of petroleum ether. As the sample moved through the column, the pigments in the plant extract separated into individual colored bands. Once the pigments were adequately separated, the calcium carbonate was removed from the column, sectioned, and the pigments recovered by extraction. Tswett named the technique chromatography, combining the Greek words for “color” and “to write.” There was little interest in Tswett’s technique until 1931 when chromatography was reintroduced as an analytical technique for biochemical separations. Pioneering work by Martin and Synge in 1941 established the importance of liquid–liquid partition chromatography and led to the development of a theory for chromatographic separations; they were awarded the 1952 Nobel Prize in chemistry for this work. Since then, chromatography in its many forms has become the most important and widely used separation technique. Other separation methods, such as electrophoresis, effect a separation without the use of a stationary phase. Prof. Faten A. Nour El-Dien 11/3/2024  Chromatographic Separations We can separate the analyte and interferents by continuously passing one sample-free phase, called the mobile phase, over a second sample-free phase that remains fixed or stationary.  Chromatography is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. Prof. Faten A. Nour Eldien Classifying Analytical Separations Analytical separations may be classified in three ways: by the method of contact between the mobile phase and stationary phase; by the physical state of the mobile phase and stationary phase; or by the chemical or physical mechanism responsible for separating the sample’s constituents. Prof. Faten A. Nour El-Dien 11/3/2024 Two common approaches are used to bring the mobile phase and stationary phase into contact. In column chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure. The stationary phase is either a solid or a thin liquid film coating on a solid particulate packing material or the column’s walls. In planar chromatography the stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action. Prof. Faten A. Nour El-Dien 11/3/2024 Classification of chromatographic methods Column chromatography Planar chromatography Thin- layer Paper Electrophoreses 11/3/2024  Classification of chromatographic methods Based upon the type of Mobile Phase: Gas Chromatography Supercritical Fluid Chromatography Liquid Chromatography Prof. Faten A. Nour El-Dien 11/3/2024 Prof. Faten A. Nour El-Dien 11/3/2024  Questions Prof. Faten A. Nour El-Dien Prof. Dr. Faten A. Nour El-Dien