edited hv -,
GEORGEL. GILBERT D e w o n University Granv8ile. Ohio 43023
A Simple Demonstration of Reversible Oxygenation Nicholas K. Kildahl Worcester Polytechnic Institute Worcester. MA 01609
The importance of transition metal ions in biological systems is now widely recognized (1 and probably merits a t least cursory coverage at the freshman level. Of the many biological functions performed by metal ions, oxygen storage and transport are certainly among the most fascinating. Nature has evolved several proteins which perform one or the other of these functions, some utilizing Fe(I1) a t the active site (e.g., hemoglobin and myoglobin, the oxygen transport and storage molecules in mammals) and others utilizing Cu(1) (the hempcyanins, found in some mollusks and arthropods (2)). When dioxygen binds to such species, the integrity of the 0-0 b o d is reserved: i.e.. irreversible oxidation of the
veistble in this conte;t indicates that the-metal-dioxygen adduct readily dissociates to regenerate 0 2 under conditions of low partial pressure of Oz. In the past several years, several low MW models for hemoglobin and myoglobin have been developed which reversibly bind dioxygen under a variety of conditions of temperature and oxygen partial pressure ( 3 ) .Many of these model compounds involve Co(I1) rather than Fe(I1) a t the active site, because Co(I1) is less prone to irreversible oxidation processes than is Fe(11). The lecture demonstration described here utilizes one of these Co(I1)-containing model compounds to visually demonstrate the phenomenon of reversible oxygenation. The structure of the complex is shown below:
898
Journal of Chemical Education
*z+
m2+
J,J,
Materials 1) CoL(C10a)2.This complex may he readily prepared by literature methodS ( 4 ) ,or as summarized in the Appendix. 2) N,N-dimethylformamide(DMF) 3) Two 100-mi graduated cylinders 4) Two household flashlights 5) 1tank 0 2 , l piece rubber tubing (4 ft), 1Pasteur pipet 6) Dry ice, broken into chunks of -1 ml volume 7) 1 1000-ml beaker containing warm (60-709C) water Procedure Prior to the lecture period, dissolve -0.4-0.5 g CoL(C104)2 in 40-50 ml DMF in one of the graduated cylinders, and pour half of the resulting yellow-oPange solution into the second graduate. Loosely clamp the two cylinders side by side an a bench top, and position a flashlight behind each one. (If the demonstration is to be done later in the lecture hour, it may be wise to flush each cylinder with N2 gas, then to stopper each cylinder lightly, in order to retard slow, irreversible oxidation of the complex which occurs in DMF solution in the presence of 02.) T o initiate the demonstration, add 3 or 4 lumps of COz to ode of the graduated cylinders, a t 5-10 sec intervals, in order to cool the contents. When the solution is cold, as evidenced by the condensation of water on the outside of the cylinder, turn on the flashlights. Bubble 0 2 gas vigorously through the cooled solution, which will undergo a rapid color change to blood red. The students a t this point may readily see the contrast in color between unoxygenated CoL, in the uncooled
graduate, and oxygenated CoL. When oxygenation is complete (10-20 s of exposure to 0 2 ) ,add a couple of lumps of C 0 2 to the graduate containing the red solution and place it in the beaker of warm water. Over a period of 10-20 s, the color of the solution will revert to yellow-orange, illustrating the reversibility of the oxygenation process. At this point, remove the graduate from the water bath and place it again in front of the flashlight, to show clearly that the color has reverted to the original yellow-orange. Discussion T h e bindina of O? hv CoL is favored thermodvnamicallv by conditionsbf ~ o ~ t e k p e r a t uand r e high partLl pressure of 09, and becomes less favorable a t higher temperature and low partial pressure of 0 2 . heref fore; in ordei to insure a dramatic color change upon exposure to 02,the DMF solution of CoL is cooled substantially before introduction of oxygen. T h e use of CO2 to effect cooling, rather than submersion in an ice bath, for example, has two obvious advantages. First, the temperature of the solution can be lowered well below O0C, so that formation of a substantial concentration of the oxygen adduct is insured. Second, the CO2 vapor which is produced during the cooling process, being more dense than air, stands over the solution and protects it from exposure to air until the lecturer is ready to initiate the oxygenation process. Once oxygenation has been demonstrated and it is desired to deoxygenate the solution, solid CO2 is again added in order to flush 0 2 from the head space above the solution, thus reducing its partial pressure, and the solution is submerged in a warm water bath to decrease the equilibrium constant for dioxygen binding. The lecturer may find it convenient to discuss the demonstration using the scheme below. CoL + 02(g) yellow
low T, high PO high T,low Po2
CoL(02) AH < 0 red
Hazards P e r c h l o r a t e salts a r e potentially explosive. Although the author has never experienced an explosion with the materials discussed here, the following precautions should be taken routinely: 1) Handle the ligand salt, L4HC104, with a porcelain spatula if
possible.
2) Do not heat either solid L4HClOn or solid COL(CIO~)~. Strong heating of solutions in organic solvents should also be avoided. 3) Do not contact either material with a strong reducing agent.
Appendix: Synthetic Procedures (abbreu L. Synthesis of ( M e s [ 1 4 ] 4 , 1 1 - d i e n e N 4 ) . 2 H m 2HC104): Ethylenediamine (17.8 ml) is dissolved in 400 ml of reagent grade acetone in a 1000-ml beaker. Next, 70% HCIO4 (28.5 ml) is added dropwise from an addition funnel, over a 112-hr period, with stirring. (Addition of HC104 should be done in a fume hood, with the door closed.) A yellow-orange color develops in the solution, followed by formation of white crystals of the macrocyclic ligand perchlorate salt, shown below.
After the mixture has cooled to room temperature (about 1 hr), the product is isolated by filtration, thoroughly washed with acetone, sucked dry, and transferred to a vial. The filtrate should he poured slowly down a sink in a fume hood, and flushed down with plenty of water Yield: >10 g. ~)~. (80 ml) is placed in a Synthesis of C O L ( C ~ O Methanol 500-ml round-bottom, three-necked flask and brought to reflux for % hr under nitrogen atmosphere. Cobalt acetate tetrahydrate (4.98 g, 0.02 mol) is added to the hot methanol, resulting in a pink-violet solution. L.2HClO.q (9.62 g, 0.02 mol) is added to the hot mixture. The resulting mixture is stirred a t reflux for 1.75 hr, during which time orange crystals of product form. The mixture is allowed to cool for 1hr, without stirring. The orange crystalline product is quickly isolated by filtration, washed once with a small portion of ethanol, sucked dry, and transferred to a vial (filtration and washing within an Ne-filled glove hag is desirable but not absolutely necessary). Yield: >7 g. The synthesis can be either scaled down or scaled up according to the wishes of the demonstrator. Literature Cited (1) "Inorganic Bixhemetry: Vol. 1: (Senior Reprrilsi: H. A. 0. Hill). American Chemical Society, 1979. (2) Senozan.N. M., J.CHEM.RDUC.,53,684 (1976). (3) Jones. R. D..Surnmervillo,D. A.,andBnsoln, F., Chum. Reu.,79.139(1979). (4) Coedkin. Y. L..Kildsh1.N. K.. and Rusch, D. H. J. Coord. Chum., 7.89 119771.
Volume GO
Number 10 October 1983
899
