Organic compounds are seldom pure. When isolated from natural sources or prepared by organic reactions, they are usually contaminated with small amounts of other compounds. Before carrying out the qualitative and quantitative analysis of organic compounds that is needed to characterize them, it is very important of purify them. Purification is a critical step in drug manufacturing, helping to eliminate unwanted materials that can be hazardous or compromise drug efficacy.
While impurities are a fact of life, the challenge is in identifying them and then performing purification processes to eliminate as many as possible. The most common way to purify solids is by crystallization, which involves using a solvent in which the solid to be purified has low solubility at room temperature but is very soluble at elevated temperatures.
If the compound does not dissolve completely in the boiling solvent, heat another portion of solvent to boiling. Add the boiling solvent dropwise to the test tube until the solid dissolves completely or until the test tube contains 3 mL of solvent. If the solid still does not dissolve, then its solubility in this solvent is too low.
Confirm that impurities are either insoluble in the hot solvent so they can be filtered out after dissolution or soluble in the cold solvent so they remain in solution after recrystallization is complete. If a solvent meets all criteria, it is suitable for recrystallization. To start recrystallization, heat the solvent to boiling on a hot plate in an Erlenmeyer flask with a stir bar.
Place the compound to be recrystallized in another Erlenmeyer flask at room temperature. Next, add a small portion of hot solvent to the compound. Swirl the mixture in the flask and then place it on the hot plate as well.
Repeat this process until the sample has completely dissolved or until addition of solvent causes no further dissolution.
Filter the solution to remove insoluble impurities. If crystals form during filtration, dissolve them with drops of hot solvent. Cool the solution on the benchtop. Cover the flask to prevent solvent loss to evaporation and to keep particulates out of the solution.
Leave the flask undisturbed until it has cooled to room temperature. Agitation during cooling may cause rapid crystallization, yielding less pure crystals. If no crystal formation is evident upon cooling, induce crystallization by gently scratching the inside walls of the flask with a glass rod or adding a small seed crystal of the compound being recrystallized.
If crystal formation cannot be induced, reheat the solution to boil off some of the solvent, and then cool the solvent to room temperature once more. Once crystals have formed, prepare an ice bath. Keeping the solution covered, cool the solution in the ice bath until crystallization appears to be complete. Clamp a filtration flask to a ring stand and connect the flask to a vacuum line. Pour the mixture of solution and crystals into the funnel and begin vacuum filtration.
Rinse any crystals remaining in the flask into the funnel with cold solvent. Wash the crystals on the funnel with cold solvent to remove soluble impurities. Continue drawing air through the funnel to dry the crystals and then turn off the vacuum pump. If necessary, the crystals may be allowed to stand at room temperature to air dry or placed in a desiccator before storing the crystallized solid.
The yellow impurities present in the crude compound have been removed, yielding an off-white solid. Based on the identity of the compound and the impurities, the purity of the crystals can be verified by NMR spectroscopy, melting point measurements, or visual inspection.
X-ray crystallography is a powerful characterization technique that identifies the three-dimensional atomic structure of a molecule. This requires a pure single crystal, which is obtained by recrystallization. Some classes of molecules such as proteins are difficult to crystallize, but their structures are extremely important for understanding their chemical functions.
With careful selection of recrystallization conditions, even these classes of molecules can be analyzed by X-ray crystallography. To learn more about this process, see this collection's video on growing crystals for crystallography. Impure reactants can cause unwanted side reactions.
Purifying reactants by recrystallization improves product purity and yield. Once a solid product has been isolated and washed, reaction yield can also be increased by removing volatiles from the filtrate and recrystallizing the product from the resulting solid.
Antifreeze proteins, or AFPs, are expressed in many organisms that live in icy environments. AFPs hinder internal ice growth by binding to ice planes, inhibiting recrystallization into larger ice crystals. Different AFPs bind to different types of ice crystal planes. Investigating AFP binding mechanisms involves adsorbing them onto single ice crystals. Proper growth of a single ice crystal is essential for clear and informative results. These proteins have applications from the engineering of cold-resistant crops to cryosurgery.
You've just watched JoVE's introduction to purifying compounds by recrystallization. You should now be familiar with the principles of the technique, a purification procedure, and some applications of recrystallization in chemistry.
An example of the results of recrystallization is shown in Figure 2. The yellow impurities present in the crude compound have been removed, and the pure product is left as an off-white solid. The purity of the recrystallized compound can now be verified by nuclear magnetic resonance NMR spectroscopy or, if it is a compound with a published melting point, by how similar its melting point is to the literature melting point.
If necessary, multiple recrystallizations can be performed until the purity is acceptably high. Seperation and purification techniques 1. The filtrate is heated over a water bath so that the vapors of the solvent may not catch fire. The contents of the flask are heated until the solid dissolves. Then the solution is cooled. A more pure solid separates out, leaving impurities dissolved in the solvent. Example for recrystallisation: 1. Add a little water and boil Some contaminants will not dissolve Transfer to a conical flask and cool Filter the crystals and allow to dry for mins Impure benzoic acid Benzoic acid after recrystallisation 7.
It is widely used for drying ethers. It finds extensive application in purifying organic chemical substances by the removal of water and carbonyl containing impurities. Use of drying agents and their properties S. It has large water absorption capacity giving the hexahydrate. Naphthalene, benzoic acid, anthracene, camphor. On cooling the vapours the solid is directly obtained. SublimationSublimation A mixture of two compounds can be separated by sublimation Simple Distillation Simple distillation is designed to evaporate a volatile liquid from a solution of non-volatile substances; the vapor is then condensed in the water condenser and collected in the receiver.
For fractional distillation, a suitable fractionating column is placed between the flask and the condenser. Fractional distillation Example: Glycerol The boiling point of a liquid is the temperature at which the total vapour pressure is equal to the external pressure. This means that by lowering the pressure the boiling point of the liquid can be lowered.
All that we have to do is to create a partial vaccum. Under reduced pressure, the substance boils at a much lower temperature and distils over undecomposed. Distillation under reduced pressure Liquid-liquid extraction requires two immiscible liquids known as the organic phase and the aqueous phase. The aqueous phase is water-based and the organic phase is an organic solvent.
Spectra are the result of searches for such absorptions over a range of wavelengths. If one determines and plots the degree of absorption by a monoatomic gas, a series of very sharp absorption bands or lines are observed. The lines are sharp because they correspond to specific changes in electronic configuration without complication from other possible energy changes. In addition to the energies associated with molecular electronic states, there is kinetic energy associated with vibrational and rotational motions.
At that time, the potential of infrared spectroscopy as an analytical tool began to be recognized by organic chemists. The change was due largely to the production of small, quite rugged infrared spectrophotometers and instruments of this kind now are virtually indispensable for chemical analysis. A brief description of the principles and practice of this spectroscopic method is the topic of this section 9. The experimental arrangement for Raman spectra is quite simple in principle.
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