CHAPTER 2 CHEMICAL METHODS USED IN PURIFICATION

GENERAL REMARKS

Greater selectivity in purification can often be achieved by making use of differences in chemical properties between the substance to be purified and the contaminants. Unwanted metal ions may be removed by precipitation in the presence of a collector (see below). Sodium borohydride and other metal hydrides transform organic peroxides and carbonyl-containing impurities such as aldehydes and ketones in alcohols and ethers. Many classes of organic chemicals can be purified by conversion into suitable derivatives, followed by regeneration. This chapter describes relevant procedures.

REMOVAL OF TRACES OF METALS FROM REAGENTS

METAL IMPURITIES

The presence of metal contaminants in reagents may sometimes affect the chemical or biochemical outcomes of an experiment. In these cases, it is necessary to purify the reagents used. Metal (and other) impurities can be determined qualitatively and quantitatively by atomic absorption spectroscopy (AAA), x-ray photoelectron spectroscopy (XPS), various mass spectrometric methods and/or inductively coupled plasma mass spectrometry (ICP-MS) (see Chapter 1, Question of Purity) and the required purification procedures can be formulated. Metal impurities in organic compounds are usually in the form of ionic salts or complexes with organic compounds and very rarely in the form of free metal. If they are present in the latter form then they can be removed by crystallising the organic compound (whereby the insoluble metal can be removed by filtration), or by distillation in which case the metal remains behind with the residue in the distilling flask. If the impurities are in the ionic or complex forms, then extraction of the organic compound in a suitable organic solvent with aqueous acidic or alkaline solutions will reduce their concentration to acceptable levels.

When the metal impurities are present in inorganic compounds as in metals or metal salts, then advantage of the differences in chemical properties should be taken. Properties of the impurities like the solubility, the solubility product (product of the metal ion and the counter-ion concentrations), the stability constants of the metal complexes with organic complexing agents and their solubilities in organic solvents should be considered. Alternatively the impurities can be masked by the addition of complexing agents which could lower the concentration of the metal ion impurities to such low levels that they would not interfere with the main compound (see complexation below). Specific procedures and examples are provided below.

DISTILLATION

Reagents such as water, ammonia, hydrochloric acid, nitric acid, perchloric acid, and sulfuric acid can be purified via distillation (preferably under reduced pressure and particularly with perchloric acid) using an all-glass still. Isothermal distillation is convenient for ammonia: a beaker containing concentrated ammonia is placed alongside a beaker of distilled water for several days in an empty desiccator so that some of the ammonia distils over into the water. The redistilled ammonia should be kept in polyethylene or paraffin-waxed bottles. Hydrochloric acid can be purified in the same way. To ensure the absence of metal contaminants from some salts (e.g. ammonium acetate), it may be more expedient to synthesise the salts using distilled components rather than to attempt to purify the salts themselves.

SCAVENGER RESINS AND OTHER SUPPORTS

There is now an extensive range of supported reactants that use resins, silica, carbons etc, to clean up reactions prior to final purification and is gaining favour in the laboratory. [See section on “Scavenger Resins” in Chapter 3, at the ends of the sections on “Preparation of other adsorbents”, “FPLC” and “HPLC”.]

USE OF ION-EXCHANGE RESINS

Application of ion-exchange columns has greatly facilitated the removal of heavy metal ions such as Cu2+, Zn2+ and Pb2+ from aqueous solutions of many reagents. Thus, sodium salts and sodium hydroxide can be purified by passage through a column of a cation-exchange resin in its sodium form, prepared by washing the resin with 0.1M aqueous NaOH then washing with water until the pH of the effluent is ~7. Similarly, for acids, a resin in its H+ form [prepared by washing the column with 0.1M aqueous mineral acid (HCl, H2SO4) followed by thorough washing with water until the effluent has pH ~7 is used]. In some cases, where metals form anionic complexes, they can be removed by passage through an anion-exchange resin. Iron in hydrochloric acid solution can be removed in this way.

Ion-exchange resins are also useful for demineralising biochemical preparations such as proteins. Removal of metal ions from protein solutions using polystyrene-based resins, however, may lead to protein denaturation. This difficulty may be avoided by using a weakly acidic cation exchanger such as Bio-Rex 70.

Heavy metal contamination of pH buffers can be removed by passage of the solutions through a Chelex X-100 column. For example when a solution of 0.02M HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] containing 0.2M KCl (1L, pH 7.5) alone or with calmodulin, is passed through a column of Chelex X-100 (~60g) in the K+ form, the level of Ca2+ ions falls to less than 2 x 10-7 M as shown by atomic absorption spectroscopy. Such solutions should be stored in polyethylene containers that have been washed with boiling deionised water (5minutes) and rinsed several times with deionised water. TES [N,N,N′,N′-Tetraethylsulfamide] and TRIS [Tris-(hydroxymethyl)aminomethane] have been similarly decontaminated from metal ions.

Water, with very low concentrations of ionic impurities (and approaching conductivity standards), is very readily obtained by percolation through alternate columns of cation- and anion-exchange resins, or through a mixed-bed resin, and many commercial devices are available for this purpose. For some applications, this method is unsatisfactory because the final deionised water may contain traces of organic material after passage through the columns. However, organic matter can be removed by using yet another special column in series for this purpose (see Milli Q water preparation, Millipore Corpn., <www.millipore.com>).

PRECIPITATION

In removing traces of impurities by precipitation, it is necessary to include a material to act as a collector of the precipitated substance so as to facilitate its removal by filtration or decantation. The following are a few examples:

Removal of lead contaminants

Aqueous hydrofluoric acid can be freed from lead by adding 1mL of 10% strontium chloride per 100mL of acid, lead being co-precipitated as lead fluoride with the strontium fluoride. If the hydrofluoric acid is decanted from the precipitate and the process repeated, the final lead content in the acid is less than 0.003ppm. Similarly, lead can be precipitated from a nearly saturated sodium carbonate solution by adding 10% strontium chloride dropwise (1-2mL per 100mL) followed by filtration. (If the sodium carbonate is required as a solid, the solution can be evaporated to dryness in a platinum dish.) Removal of lead from potassium chloride uses precipitation as lead sulfide by bubbling H2S, followed, after filtration, by evaporation and recrystallisation of the potassium chloride.

Removal of iron contaminants

Iron contaminants have been removed from potassium thiocyanate solutions by adding a slight excess of an aluminium salt, then precipitating aluminum and iron as their hydroxides by adding a few drops of ammonia. Iron is also carried down on the hydrated manganese dioxide precipitate formed in cadmium chloride or cadmium sulfate solutions by adding 0.5% aqueous potassium permanganate (0.5mL per 100mL of solution), sufficient ammonia to give a slight precipitate, and 1mL of ethanol. The solution is heated to boiling to coagulate the precipitate, then filtered. Ferrous ion can be removed from copper solutions by adding some hydrogen peroxide to the solution to oxidise the iron, followed by precipitation of ferric hydroxide by adding a small amount of sodium hydroxide.

Removal of other metal contaminants

Traces of calcium can be removed from solutions of sodium salts by precipitation at pH 9.5-10 as the 8-hydroxyquinolinate. The excess 8-hydroxyquinoline acts as a collector and is extracted out with an organic solvent.

EXTRACTION

In some cases, a simple solvent extraction is sufficient to remove a particular impurity. For example, traces of gallium can be removed from titanous chloride in hydrochloric acid by extraction with diisopropyl ether. Similarly, ferric chloride can be removed from aluminium chloride solutions containing hydrochloric acid by extraction with diethyl ether. Usually, however, it is necessary to extract an undesired metal with an organic solvent in the presence of a suitable complexing agent such as dithizone (diphenylthiocarbazone) or sodium diethyl dithiocarbamate. When the former is used, weakly alkaline solutions of the substance containing the metal impurity are extracted with dithizone in chloroform (at about 25mg/L of chloroform) or carbon tetrachloride until the colour of some fresh dithizone solution remains unchanged after shaking. Dithizone complexes metals more strongly in weakly alkaline solutions. Excess dithizone in the aqueous medium is removed by extracting with the pure solvent (chloroform or carbon tetrachloride), the last traces of which, in turn, are removed by aeration. This method...