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. Figure 2. Recrystallization is a method of purifying a compound by removing any impurities that might be mixed with it. It works best when the compound is very soluble in a hot solvent, but very insoluble in the cold version of the same solvent. The compound must be a solid at room temperature. Recrystallization is often used as a final clean-up step, after other methods such as extraction or column chromatography that are effective at removing larger amounts of impurities, but that do not raise the purity of the final compound to a sufficiently high level.
Recrystallization is the only technique that can produce absolutely pure, perfect single crystals of a compound. These crystals can be used for X-ray analysis, which is the ultimate authority in determining the structure and three-dimensional shape of a molecule.
In these cases, the recrystallization is allowed to proceed very slowly, over the course of weeks to months, to allow the crystal lattice to form without the inclusion of any impurities. Special glassware is needed to allow the solvent to evaporate as slowly as possible during this time, or to allow the solvent to very slowly mix with another solvent in which the compound is insoluble called antisolvent addition.
The pharmaceutical industry also makes heavy use of recrystallization, since it is a means of purification more easily scaled up than column chromatography. These different crystal forms might have different biological properties or be absorbed into the body at different rates.
A more common use of recrystallization is in making rock candy. Rock candy is made by dissolving sugar in hot water to the point of saturation. Wooden sticks are placed into the solution and the solution is allowed to cool and evaporate slowly. After several days, large crystals of sugar have grown all over the wooden sticks. Mayo, D. Armarego, W. Ray, P. Google Patents: Hightower, T. Journal of Chemical Education 83 11 , Rohani, S. Organic Chemistry. Purifying Compounds by Recrystallization.
To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Login processing Previous Video Next Video. Overview Source: Laboratory of Dr. Jimmy Franco - Merrimack College Recrystallization is a technique used to purify solid compounds.
Perform all steps in a fume hood to prevent exposure to solvent fumes. Selecting a Solvent Place 50 mg of the sample N-bromosuccinimide in an Erlenmeyer flask. If the sample dissolves completely, the solubility in the cold solvent is too high to be a good recrystallization solvent. If the sample does not dissolve in the cold solvent, heat the test tube until the solvent boils.
If the sample has not completely dissolved at this point, add more boiling solvent drop-wise, until all of the solid dissolves. If it takes more than 3 mL to dissolve the sample in the hot solvent, the solubility in this solvent is probably too low to make it a good recrystallization solvent. If the first choice of solvent is not a good recrystallization solvent, try others.
If a single solvent that works cannot be found, try a two solvent system. If you cannot find a suitable single solvent system, then a solvent pair may be necessary. When identifying a solvent pair, there are several key considerations 1 The first solvent should readily dissolve the solid.
As a general rule "likes dissolve likes" meaning that polar compounds tend to be soluble in polar solvents and non-polar compounds are often more soluble non-polar compounds. Also make sure the boiling point of the solvent is lower than the melting point of the compound, so the compound forms as solid crystals rather than as an insoluble oil.
Confirm that the impurities are either insoluble in the hot solvent so they can be hot-filtered out, once the compound is dissolved or soluble in the cold solvent so they stay dissolved during the entire process. This is a better choice than a beaker, since the sloping sides help trap solvent vapors and slow the rate of evaporation.
In this case, the hot solution with the solute dissolved completely in the hot solvent must be filtered while it is hot, without allowing it to cool more than a few degrees to prevent any re-crystallization to take place. An impure compound is dissolved the impurities must also be soluble in the solvent , to prepare a highly concentrated solution at a high temperature.
The solution is cooled. Decreasing the temperature causes the solubility of the impurities in the solution and the substance being purified to decrease. This second series of diagrams shows what happens if you cool the solution too quickly.
The yellow triangle impurities are trapped inside the crystals being formed by the orange hexagons, thus, the crystals isolated are impure. It is very easy to get a precipitate, but very difficult to get crystals. We already mentioned temperature and time being key factors for successful recrystallization. Precipitation typically occurs when the temperature has not been lowered gradually. Furthermore, we must not disturb the solution or its container as it cools even though it is tempting.
Disturbing the solution can break up any seed crystals 6 that have started growing. Such crystals are often referred to as single crystals, and not only must they be completely pure, but also the crystal lattice and growth must be highly ordered. This task can be monumental, as very small variables can be detrimental to the growth of a single crystals.
During grad-school, a post-doc told me to leave the crystals growing in a room where nobody ever went, as even the vibrations of footsteps or laboratory equipment could be detrimental. Please also note that some compounds simpl crystallize more easily than others.
More rigid molecules are, as a rule, easier to crystalize. Let us go through a recrystallization process, focusing on technical aspects and trouble shooting. Typical problems: Adding too much solvent so that the product does not crystallize later.
Typical problems: Crystals do not form at all too much solvent , precipitate forms instead of crystals temperature has dropped too quickly, or an oil forms.
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