Ind. Eng. Chem. Res. 1995,34, 2553-2654
2553
Ligand-Exchange Chromatography: A Brief Review Harold F. Walton Cooperative Institute for Research in Environmental Sciences, University of Colorado at Boulder, Boulder, Colorado 80309-0216
One of Fred Helfferich’s outstanding contributions to the science of chemical separations is ligand-exchange chromatography. He first described this method in a letter to Nature in 1961. He wrote, “The new technique combines two fields of chemistry, namely, ion exchange and coordination chemistry, in order to accomplish a task that neither could do alone.” To honor Professor Helfferich I present a very brief review of ligand exchange and some of its applications. In ligand-exchange chromatography the stationary phase is an ion exchanger that carriers metal ions, generally those of a transition metal, that can bind molecules of the substances to be separated, the ligands. The ligands are electron-donor molecules, usually amines, amino acids, or hydroxy compounds. They are carried in the mobile phase, along with a displacing ligand which is usually ammonia, but may be water itself, the solvent. While the ligands move with the solvent, the metal ions remain stationary, attached to the ion exchanger. Of course this is an idealization; the metal ions do move to some extent, displaced by ammonium ions or other protonated ligands. In a typical ligand-exchange separation a column is packed with a cation-exchangeresin, and then a solution of cupric sulfate in aqueous ammonia is passed. The resin becomes loaded with cupric ions in the form of their ammonia complexes. Then dilute aqueous ammonia is passed t o wash out the excess of copperammonia ions. Copper(I1)ions are chosen because they form by far the most stable M(I1) complexes of the first series of transition elements. A dilute solution of ammonia is passed as the mobile phase, and the mixture to be separated, say two amines, ANH2 and BNH2, is introduced a t the column inlet. Both these amines travel down the column, becoming attached to the stationary copper ions and displaced by the ammonia until they emerge at the column exit. The amine that forms the weaker complex with copper(I1) comes out first; the amine that forms the stronger complex comes out later. Helfferich conceived the new method when he was faced with the task of separating and recovering 1,3diamino-2-propanolfrom a dilute aqueous solution that also contained ammonia. He passed the solution through a glass column packed with a carboxylic cation-exchange resin loaded with copper-ammonia complex ions. He chose the carboxylate resin because the functional -COO- ions hold copper ions very strongly. The diamine was absorbed in preference to ammonia, and became visible on the resin because of the intense blue color of its copper complex. The 1,2- and 1,3-diamines form very stable metal complexes because of the chelate effect, and they are strongly absorbed from dilute solutions. Nevertheless the diamine can be stripped off the column, and recovered in concentrated form, by simply passing a concentrated ammonia solution. Two ammonia molecules displace one diamine molecule; therefore, the equilibrium Cu(NH,),
+ diamine = Cddiamine) + 2NH3
is displaced t o the left by increasing the concentration. This is the key to the successful recovery of the diamine. The situation is analogous to that in water softening, where calcium ions displace sodium ions from the exchanger in dilute solution and are then displaced from the exchanger by sodium ions when the bed is “regenerated’’ by passing a concentrated sodium chloride brine. Ligand-exchange separations should be very versatile. One can vary the nature of the ion exchanger, both in the polymeric “backbonenand in the functional group. One can vary the metal ion and the eluent. Early studies showed that elution orders of different amines could be manipulated almost at will by changing the type of exchanger and the metal ion. Nevertheless, some problems arise that have prevented full exploitation of this versatility. One problem is that one cannot conveniently use ligand exchange in the mode of highperformance ion chromatography. The concentration of the displacing ligand must be low to match the low ionexchanging capacity of the column packing, but the water concentration is as high as ever, and water acts as a competitive displacing ligand, precipitating copper hydroxide. Another problem is that the metal ions do not remain immobile; they are always displaced by the eluent to some extent, Displacement can be minimized by using an ion exchanger with functional chelating groups, like the iminodiacetate groups of the commercial resin Chelex-100,but these tridentate groups reduce the coordinating capacity of the metal ions and reduce the swelling of the resin, so that ligand exchange is slow. Resins with functional sulfonate ions have good chemical and mechanical properties, but they do not hold metal ions as strongly as carboxylate or chelating resins. One can compensate for the weak retention of metal ions by adding metal salt to the aqueous ammonia eluent, thus maintaining a constant metal-ion concentration in the mobile phase, but the very dilute metal salt solutions are slow to reach equilibrium with the resin and are therefore difficult to control. At one time, ligandexchange chromatography seemed a promising way t o analyze amino acid mixtures, and commercial instruments were built, but they were not a success, because it was too hard to control the metal-ion concentrations. The presence of metal ions in the effluent is a nuisance in preparative chromatography, but in analytical chromatography it may be an advantage. The metal ions are present as coordination complexes with the ligands, and transition-metal complexes show characteristic light absorption in the visible and ultraviolet. Non-light-absorbing ligands may therefore be detected by ultraviolet absorption of their metal complexes, which is a great help. This possibility has not been exploited in ligand-exchange chromatography as much as it should. Today, two applications of ligand-exchange chromatography stand out for their practical value. They are very different. One is the analysis of mixtures of sugars and other carbohydrates; the other is the separation on the preparative scale of enantiomers of amino acids. The first uses as the stationary phase a sulfonated polysty-
Q888-5885/95/2634-2553$09.QQ/Q0 1995 American Chemical Society
2554 Ind. Eng. Chem. Res., Vol. 34, No. 8,1995
rene ion exchanger carrying calcium ions; the eluent, and the displacing ligand, is pure water. The second, developed to a high degree by Vadim Davankov, uses a special styrene polymer with an open structure which carries, grafted to the polymer structure, an optically active amino acid, generally L-proline, which in its turn is coordinated with copper(I1)ions. The copper ions can bind a second amino acid molecule from the mobile phase, and the binding is much stronger for one amino acid enantiomer than for the other. Separation factors up to 4 can be obtained. The mobile phase is aqueous ammonia. A variant of this method that gives excellent chromatography of chiral amino acid mixtures uses ligand exchange in the mobile phase. The mobile pase contains the binary copper complex of a single chiral form, say D-phenylalanine. The stationary phase is nonpolar, say c 1 8 silica. It retains uncharged CuLlLz but not ionic species. When a DL mixture is introduced into the column, the chiral form giving the most stable binary complex is retained longest. It is interesting to note the totally different chemistries of the two methods. In the first, calcium-ligand complexes are formed that have formation constants of the order of 1-4. They are extremely weak, compared with the copper-amino acid complexes, which have formation constants about lo8, referred to the anions of the amino acids. In both cases, and indeed in all applications of ligand exchange, stereochemistry is of major importance. Bindig of sugar molecules to calcium ions is stronger, the more coordinate links can be formed at once, and this in turn depends on the orientation of the hydroxyl groups in the ring structure of the sugar. Goulding showed that the axial-equatorial-axial configuration allowed three coordinate links to be formed between a sugar molecule and a calcium ion, leading to strong binding. The most important commercial application of ligand exchange is the separation of fructose from glucose in corn syrup t o give high-fructose syrup. A calciumloaded ion exchanger is used, and while this is usually
a sulfonated polystyrene resin, inorganic exchangers (artificial zeolites) are used as well. Fructose is held about twice as strongly as glucose. Countercurrent, continuous flow systems have been devised. One other application of ligand-exchange chromatog raphy deserves special mention for its practical value; this is immobilized metal afinity chromatography, developed by Jerker Porath. It is used to separate proteins. The stationary phase is agarose carrying chelating groups such as EDTA. These in turn carry coordinated metal ions. The agarose structure is hydrophilic, allowing fast diffusion of protein molecules in and out of the exchanger. (We noted above that polystyrene-based chelating exchangers carrying metal ions do not swell enough in water to allow fast exchange.) Many more illustrations could be cited, but these are enough to show that Fred’s vision has borne fruit.
Literature Cited (1) Helfferich, F. Ligand Exchange: a novel separations technique. Nature 1961, 189, 1001. (2) Helfferich, F. Ligand exchange; (a) Equilibrium, (b) Separation of ligands having different coordination valencies. J . Am. Chem. SOC.1962,84, 3237, 3242. (3) Davankov, V. A.; Navratil, J. D.; Walton, H. F. Ligand Exchange Chromatography; CRC Press: Boca Raton, FL, 1988. (4) Walton, H. F.; Rocklin, R. D. Zon Exchange in Analytical chemistry; CRC Press: Boca Raton, FL, 1990. (5) Goulding, R. W. Liquid chromatography of sugars and related polyhydric alcohols on cation exchangers: effect of cation variation. J . Chromatogr. 1975, 103, 229. (6) Wortel, Th.M.; van Bekkum, H. Carbohydrate separation by X-zeolites. Cation and solvent effects. Red. Trav. Chim. 1978, 97, 156. (7) Porath, J.;Olin, B. Immobilized metal ion affinity adsorption and affinity chromatography of biomolecules. Biochemistry 1983, 22, 1621.
Received for review October 17, 1994 Revised manuscript received December 9,1994 Accepted January 17, 1995
IE940603H
