The purification of substances is one of the most commonly encountered difficulties in the chemical manufacturing industry. Purification may be necessary not only for the end product that is marketed to consumers, but may also be necessary for intermediates that are produced in the course of making the end product from simpler starting materials. As a result, the manner in which purifications are conducted, and their efficacy in accomplishing purification at satisfactory level, may have a substantial economic impact on the production of chemical substances. A diverse array of methods have been employed for this purpose.
Purification by Extraction
It is sometimes the case that the substance(s) of interest, i.e. target(s), in a separation may substantially differ in their solubility characteristics from the other, undesired substances present in the mixture i.e., interferents/impurities, to such a degree that they may be purified by simple extraction procedures. For example, a compound that is largely insoluble in water may be separated from water soluble interferents/impurities by shaking the mixture with water and a water immiscible solvent. After allowing the two immiscible phases to separate, the interferents/impurities will be in the aqueous phase, while target(s) will be in the water immiscible phase. Physical separation of the two phases and subsequent evaporation of the water immiscible solvent will yield the target(s).
In another example, target(s) that exhibit significant degrees of acid or basic properties can be separated in a variant of this procedure that involves the temporary modification of the charge state of a target(s) by appropriate modification of the pH of the aqueous phase. The change in charge state may convert neutral water insoluble target(s) to water soluble species that can be separated by extraction from neutral interferents/impurities. A subsequent change in the aqueous pH to return to neutral water insoluble target(s) allows their separation from water soluble interferents/impurities through an additional extractive procedure.
Though the extractive procedures described above are amongst the most powerful and inexpensive tools that may be used in the purification of substances they typically suffer from a lack of specificity. In particular, these extractive methods will not separate substances that share similar solubility properties. Thus, procedures of this type are often referred to as “group separations” in which groups of compounds having similar gross solubility properties are separated from other groups of compounds having differing gross solubility properties.
Purification of Substances by Distillation and Crystallization
Fractional or simple distillation are amongst the most effective and least expensive methods that can be employed for the purification of appropriate substances. However, this technique can not be employed for substances having low vapor pressures or that are thermally unstable. Many substances fall into this category, and so distillation is used primarily for the purification of the simple, low molecular substances that serve as the ultimate starting materials for the synthesis of compounds of greater economic interest.
Crystallization may also be an extremely effective method for purification of some substances. It is well suited to the purification of large amounts of material and can be easily performed. However, many substances do not crystallize readily. In many instances the crystallization of a substance may be adversely affected by the presence of significant quantities of interferents/impurities. Since this is a commonly encountered situation in many chemical syntheses, the use of crystallization in industrial operations, while extremely important for many chemical manufacturing processes, cannot be said to be of extremely broad utility and reliability.
Purification of Substances by Derivatization
One of the means by which difficulties in crystallization or separation by other means may be circumvented is the use of chemical derivatization. In this procedure, a target may be subjected to a chemical reaction with some co-reactant to convert it to new substance i.e., a derivative, that has substantially different physical properties. These different properties may be solubility, or susceptibility to crystallization, or some other property. After separation of a derivative from interferents/impurities, another chemical reaction is applied to the derivative to convert it back to the target.
Though occasionally employed, this procedure may suffer from disadvantages. For example, there are few chemical reactions that proceed in 100% yield. Since two chemical reactions are involved in this overall process (formation of a derivative and reconversion back to a target) decreases in overall yield of purified target are likely. The process may also be costly from a number of standpoints: the chemical reactions involved require some amount of time, and the reagents and derivatizing agents have associated expenses. The latter often represents a particular problem since it is rare that the process of returning to a target from a derivative yields the derivatizing agent in a form that can be reutilized directly for other purifications.
Purification of Substances by Chromatography
Probably the most versatile method for the purification of a wide variety of chemical substances is chromatography. Most chromatography methods separate substances on the basis of their differential affinities for a stationary phase and a mobile phase. These two phases may both be liquid (e.g., as for countercurrent chromatography), but the combinations in most common use are gas with liquid (gas liquid chromatography, GLC or GC) and liquid with solid. The latter category is that which is most commonly employed for the purification of significant quantities of substances. The basic principles of chromatography are the same for all of these methods.
In one version of liquid-solid chromatography, a mixture of substances is applied (often in the form of a solution) to the top of a column that contains a granular or powdered solid adsorbent (e.g., silica gel, alumina). The mixture is then eluted by passing an appropriate solvent (the eluent) through the column and collecting it in portions at the bottom of the column. It is during the process of elution that separation of the substances occurs, and the cause of the separation is the differential affinities of the substances for the solid phase and the liquid phase (the eluent). For example, consider substances A and B. in which A has a much stronger affinity for the solid phase than the liquid phase, and B has a much stronger affinity for the liquid phase than the solid phase. As the eluent flows through the column, both substances will be in equilibrium between the solid phase and the liquid phase; that is, each substance will spend part of the time dissolved in the liquid phase and part of the time adsorbed onto the solid phase. In the example presented, A will spend most of its time adsorbed to the solid, while B will spend most of its time in the liquid. The solid is not moving through the column (i.e., is the stationary phase), and since A is usually (but not always) adsorbed to the solid, it will move through the column very slowly. On the other hand, B will move through the column very rapidly, since it will generally be in the mobile liquid phase. Because of their different rates of travel through the column, A and B will thus be physically separated.
Since it is the differences in affinities that substances have for the solid and liquid phases that is responsible for their different rates of travel through a column (and hence their separation), it is clear that the nature of these affinities is critical to the separation process. Typically, the affinity of a substance for a solid or liquid phase is due to the sum of a large number of weak intermolecular forces. These may include ion-dipole interactions (amongst the strongest of which is hydrogen bonding), charge-charge interactions, and hydrophobic interactions. The three dimensional disposition of functional groups in a molecule, along with the propensity of each of these functional groups to engage in these types of interactions with either the stationary or mobile phases are what determine the rate of travel in the elution process.
Problems with Chromatography
There are, however, some practical problems with the chromatographic separation of substances that arise as a consequence of the presence of interferents/impurities. The two general classes of interferents/impurities that may rise to problems in chromatographic separations are those that are difficult to elute and those that elute at a rate similar to a target.
Difficultly eluted interferents may, for example, be a problem when there is a desire to reuse the stationary phase for repeated separations. Stationary phases can be quite expensive, and the ability to use the column for multiple purifications may lead to a significant cost savings. For example, in order to take advantage of the reusability of a stationary phase it may be necessary that a majority of the substances in a sample applied to the column be eluted prior to applying a second batch of sample. Otherwise, more slowly moving substances from the first chromatography may elute in conjunction with a target in the second chromatography. Additionally, if difficultly eluted interferents are present in a sample, then large volumes of eluent may be necessary to remove them, which may result in higher costs associated with eluent purchase. In some cases, interferents may be irreversibly adsorbed to the column. In this case, it may be necessary to perform sample pretreatment to remove these irreversibly adsorbing interferents prior to loading on the column. Alternatively, a sacrificial pre-column can be placed in front of the true separation column, and then discarded when it is saturated with irreversibly adsorbing interferents. Both of these solutions, however, may be undesirable due to the costs in materials and time they entail.
Eluents that elute at a similar rate to the target may pose a more subtle problem in some circumstances. Chromatography may be performed in what has been termed the “analytical regime” or the “overload regime.” In the analytical regime, the proportions of the substances being separated to the amount of stationary phase is such that a true equilibrium between the mobile and stationary phases may be established. Within the analytical regime, the time it takes a substance to elute will not be substantially affected by amount loaded onto the column. In the overload regime, a much larger amount of the substance is applied to the column. In this case, there is not sufficient stationary phase to fully adsorb the substances present, and a true equilibrium does not exist. The result is that sample bands move faster, and they broaden, which may decrease the resolution of the separation. In other words, substances that might be separated readily with low sample loading (in the analytical regime) may not separate well when higher loading levels are employed (in the overload regime).
The change in retention behavior associated with different loading levels may not be desirable. For example, loading in the analytical range may utilize weight ratios of sample to stationary phase on the order of 1:100 or much higher, whereas the overload regime might utilize only a 1:10 ratio. Thus, in this example, for a given amount of stationary phase, one may perform a chromatography one time in the overload regime versus ten times in the analytical regime. Since roughly the same quantities of eluent are required in each case, the analytical regime separation may require a higher cost. Separations in an overload mode may therefore be desirable in some applications. For example, this may only be carried out if a good enough separation has been achieved that the band broadening and increased rate of travel associated with this process does not result in overlapping of elution bands.
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