Lecture 3: Chromatography and Separation Techniques PDF

Lecture 3 Course: Chromatography and Separation Techniques Code: PMC 322 Dr. Ahmed Sayed Saad Dr. Ahmed Sayed Saad Instrumental Analysis (PMC 227) 1  Precipitation Dr. Ahmed Sayed Saad 2 ...

Lecture 3 Course: Chromatography and Separation Techniques Code: PMC 322 Dr. Ahmed Sayed Saad Dr. Ahmed Sayed Saad Instrumental Analysis (PMC 227) 1  Precipitation Dr. Ahmed Sayed Saad 2  Precipitation Dr. Ahmed Sayed Saad 3  Separation of Species Present in Trace Amounts by Precipitation The techniques required to precipitate trace amounts differ from those used when the analyte is present in large amounts. Difficulties: 1. Solubility losses 2. Supersaturation often delays ppt formation 3. Difficult to coagulate small amounts of colloidal dispersed substances. Solution: o To minimize these difficulties, add a quantity of some other ion that also forms a precipitate with the reagent. o The precipitate from the added ion is called a collector and carries the desired minor species out of solution. Mechanism: o A collector may entrain a trace constituent as a result of similarities in their solubilities. o Other collectors function by coprecipitation in which the minor component is adsorbed on or incorporated into the collector precipitate as the result of mixed crystal formation. Dr. Ahmed Sayed Saad Instrumental Analysis (PMC 227) 4  Separation of Species Present in Trace Amounts by Precipitation Example: Isolating manganese as the sparingly soluble manganese dioxide, a small amount of iron(III) is frequently added to the analyte solution before the introduction of ammonia as the precipitating reagent. The basic iron(III) oxide(the collector) brings down even the smallest traces of the manganese dioxide. Fe3+ MnO2 Fe3+ Fe3+ Fe2O3 Mn2+ Dr. Ahmed Sayed Saad 5 Separation by Electrolytic Precipitation In this process, the more easily reduced species, either the wanted or the unwanted component of the sample, is isolated as a separate phase. (Reduction occurs at the cathode) The mercury cathode has found wide application in the removal of many metal ions prior to the analysis of the residual solution. (remember Polarography is mainly used in reduction reactions especially for metals to from amalgams) Dr. Ahmed Sayed Saad 6  Problem Should the working electrode be cathode or anode? Which ion should I start reducing first? Dr. Ahmed Sayed Saad 7  Problem Dr. Ahmed Sayed Saad 8  Salt-Induced Precipitation of Proteins A common way to separate proteins is by adding a high concentration of salt. This procedure is termed salting out the protein. The solubility of protein molecules shows a complex dependence on pH, temperature, ionic strength, the nature of the protein, and the concentration of the salt used. At low salt concentrations, solubility is usually increased with increasing salt concentration. This salting in effect is explained by the Debye-Hückel theory. The counter ions of the salt surround the protein, and the screening results in decreasing the electrostatic attraction of protein molecules for each other. This decrease, in turn, leads to increasing solubility with increasing ionic strength. Dr. Ahmed Sayed Saad 9  Salt-Induced Precipitation of Proteins At high concentrations of salt, however, the ↓repulsive effect of like charges is reduced as are the forces leading to ↓solvation of the protein. When these forces are reduced enough, the protein precipitates and salting out is observed. Ammonium sulfate is an inexpensive salt and is widely used because of its effectiveness and high inherent solubility. At high concentrations, protein solubility, S is given by the following empirical equation: log 𝑆 = 𝐶 − 𝐾𝜇 where C is a constant that is a function of pH, temperature, and the protein; K is the salting out constant that is a function of the protein and the salt used; and µ is the ionic strength. Dr. Ahmed Sayed Saad 10  Salt-Induced Precipitation of Proteins Proteins are commonly least soluble at their isoelectric points. Hence, a combination of high salt concentration and pH control is used to achieve salting out. Protein mixtures can be separated by a stepwise increase in the ionic strength. Care must be taken with some proteins because ammonium sulfate can denature the protein. Alcoholic solvents are sometimes used in place of salts. They reduce the dielectric constant and subsequently reduce solubility by lowering protein-solvent interactions. Dr. Ahmed Sayed Saad 11  Separation by Distillation Distillation is widely used to separate volatile analytes from nonvolatile interferents or vice versa. Distillation is based on differences in the boiling points of the materials in a mixture. A common example is the separation of nitrogen analytes from many other species by converting the nitrogen to ammonia, which is then distilled from basic solution. Other examples include separating carbon as carbon dioxide and sulfur as sulfur dioxide. Distillation is widely used in organic chemistry to separate components in mixtures for purification purposes. Dr. Ahmed Sayed Saad 12  Separation by Distillation There are many types of distillation: Vacuum distillation is used for compounds that have very high boiling points. Lowering the pressure to the vapor pressure of the compound of interest causes boiling at lower temperatures. Used for separating components with high boiling points, such as in petroleum refining and pharmaceuticals. Molecular distillation occurs at very low pressure (0.01 torr) such that the lowest possible temperature is used with the least damage to the distillate. Ideal for separating sensitive substances like vitamins, oils, and perfumes. Pervaporation is a method for separating mixtures by partial volatilization through a membrane. The membrane selectively allows one component to pass, which then evaporates on the other side Flash evaporation is a process in which a liquid is heated and then sent through a reduced pressure chamber. The reduction in pressure causes partial vaporization of the liquid. Dr. Ahmed Sayed Saad 13  Separation by Distillation Method Description Applications Advantages Limitations Uses reduced pressure Limited to compounds to lower boiling points, Petroleum refining, Allows distillation at stable under Vacuum Distillation preventing pharmaceuticals. lower temperatures. moderately low decomposition of heat- vacuum. sensitive compounds. A type of short-path Purification of Requires specialized vacuum distillation Ideal for heat-sensitive Molecular Distillation vitamins, essential oils, equipment for ultra- under extremely low materials. and pharmaceuticals. low pressures. pressures (