GEORGEL. GILBERT Denison University Granville. Ohio 43023
SUBM~TED BY
Rex D. Ackerson Northern Oklahoma College Tonkawa, OK 74653
CHECKEO BY
David Speckhard LOWSCollege Oubuque. IA 52001
Dry ice, freon-12 air-conditioning refrigerant, and small freezer bags may be used to demonstrate volume changes, vapor-liquid equilibrium, a simulation of rain formation, and heat of vaporization. The bag is "zipped" almost shut leaving enough opening to insert the tubing from a can of Freon-12 refrigerant. Freou-12 is available for less than $2.00 from most hardware stores and auto departments. The tubing is inserted and allowed to fill the bag, which has been previously flattened to force out most of the room air. I t is zipped shut assoon as it is moderately filled. Pieces of dry ice are placed on top of the bag and the bag is observed from the side. The bag will immediately but gradually begin to collapse as condensation of the Freon-12 occurs. This is easilv visible and and can be related to difference in volumes of liouids. The bae" will.. of course. re-inflate if the drv ice is removed due to vaporizatiun. If the piece of dry ice is the rieht size.. vou can achieve an eauilibrium situation in which " " the processes of vaporization and condensation are in equilibrium. In this situation, if the dry ice is in the center of the top of the bag, a rainfall effect can be observed as drops of Freon-12 form a t the upper surface and fall to the bottom of the bag only to vaporizeagain. Caution: Care should be used in handling dry ice due to the daneer of frostbite. Caution should also he exercised with Freon-12. It can cause frostbite, liver damage, and pressure explosions. Visibility of this demonstration is a drawback in the classroom. For maximum effectiveness the bag needs to be carried out among the students after it has been set up. Overall, this is a relatively simple demonstration to do if dry ice is available. I t relates well to weather phenomena and t o refrigeration techniques and is quite intriguing to most students.
Inorganic Fireflies-A Chemiluminescent Clock Reaction SUBM~TTED BY
Peter Jones, Jane E. Frew, and Nina Scowen Univerrlly of Newcastle upan Tyne Newcartie upon Tyne. N E l 7RU, U.K.
CHECKEO BY
Michael Beasley Carleton College Northfield, MN 55057
Clock reactions are among the most aesthetically pleasing, versatile, and dramatic chemical demonstration experiments and have long been an obsession of P.J. We have adapted the clock technique to illustrate the application t c 70
Journal of Chemical Education
X
=
H, deuteroferriheme
X
=
-CH=CHCH9, protoferriheme
Structures and nomenclature of fernihemes (iron(llltporphyr1ns). Trivial names: Heme-an iron-porphyrin Ferriheme-an iron(ll1tporphyrin Oeuterofeniheme-an iron(ll1tporphyrinwith lhe particular set of porphyrin SUbStitUentS indicated above.
flow methods in the study of fast reactions ( I ) and to illustrate kinetic problems in the release of carbon dioxide from the bloodstream in respiration (2). The endpoint of most clock reactions is marked by a sudden change in color of the reaction solution. In this experiment the endpoint is marked by a flash of light! Although a chemiluminescent clock reaction has been described previously (31, the present experiment uses dilute aqueous solutions and the chemical principles involved are related to important areas of enzyme chemistry and analytical chemistry. Chemical Prlnclples of the Reactlon
The chemiluminogenic oxidation of luminol(1) by hydrogen peroxide in aqueous alkaline solution is a well-known demonstration experiment.
In the reaction aminophthalate ion is formed in an excited state and decays to the ground state with emission of a = 424 nm, quantum yield 4 . 0 1 ) . The reaction photon (A,. is catalyzed by a variety of species of which ferriheme complexes (iron(II1)-porphyrins, see figure), heme enzymes (peroxidases, catalases), and the cytochrome derivative microperoxidase are the most effective. We have been studying the mechanism of catalytic action of ferrihemes in a variety of peroxide oxidations (4). The clock reaction involves a competition between luminol and another more efficient reducing substrate (e.g., ascorbate) (5) for active oxidizing intermediates formed in the reaction,
saturated sodium chloride solution and 2 mL concentrated HCI. Allow to stand for >12 h. Filter off the crystals and wash successively with 30mL of 50%aqueous aceticacid, 50mL distilled water, and 20 mL ether. Allow to dry in air. Typical yield >50%.
ferriheme
1+
".09
oxidizing intermediate
\ dark reaction
light
although the detailed mechanism is more complex (6).If exoerimental conditions are arranged so t h a t the amount of ascorbate is limited, light emission is observed after ascorhate oxidation is complete. Experimental Stock solutions H202-0.25 mol L-' in distilled water Carbonate buffer (pH 9) (3.86 g anhydrous NaHC03, 0.44 g anhydrous Na2CO1,25.8 g NaCl in 1 L distilled water) Luminol-5 X 10Wmd L-1 (see below) Deuteroferriheme (see figure)-2.6 X 10-'mol L-' (see below) Ascorbic aeid-in the range 1 X 10V to 3 X 10W mol L-' in distilled water Reaction Solutions Solution A: 4 mL H202,6 mL carbonate buffer Solution B: 5 mLdeuteroferriheme. 5 mL luminol, 5 mL ascorbic acid Procedure In a darkened room, stir solution B, and add solution A. The reaction time depends on the ascorbic acid coneentration, e.g., using 2 x 10-5 mol L-' ascorbate (4 X 1 0 - k o l L-' in the reaction solution) gives a reaction time of -20 s at room temperature (-20 o -,. r\
Why use deuteroferriheme (which is not commercially available)
which the light produced-is internally absorbed by the reaction solution. The waveleneths of lieht emitted in the luminol reaction overlan the most intense ahsorotion band of ferrihemes (Soret band ~~~
tuents in the pyrrole rings of the porphyrin (see figure). Deuteroferriheme has a much Lower dimerization constant than protoferriheme, so, s t the same total concentration, the coneentration of highly active monomeric ferriheme will be much greater in a deuteroferriheme solution than in a ~rotoferrihemesolution. The required rate of reaction can be achievkd with a much lower catalyst concentration using deuteroferriheme and much less of the light produced is internally absorbed. Preparation of Chlorodeuteroferriheme from Hemin Grind 2 g heminchloride and 6 g resorcinol together and heat the mix at 150-160 T (air condenser, ail bath) for -45 min. Allow the mixture to cool and solidify overnight. Grind the dark brown solid' and wash with ether until the washings are almost colorless. The crude sample of deuteroferriheme is left to dry overnight. Recrystsllize as follows: dissolve the sample in 10 mL pyridine, add 15 mL chloroform, and shake for 15 mi". Filter through a sintered glass funnel, and wash the residue with4 mLchloroform. Pour the filtrate into a boiling solution of 160 mL glacial acetic acid containing 3 mL -
-
' Do not despair if the material is black, tarry. and nongrindable at
this stage. Scrape out and commence washing with ether. The material should rapidly become dark brown and grindable.
Preparing Solutions of Deuteroferriheme and Luminol These materials dissolve readily in alkali. For deuteroferriheme, disdve the weiehedsamole in 2mLof0.1 molL-1 NaOH. andmake up wilhd~st~llerl water. For iummddiwrivr rhe we~yhedwnplrin2 ml. of I m d L-'iiaOH, and m.2kc up u,ilh diardied xatrr.
Discusdon Topics Nature of the Oxidizing Intermediates T h e mechanism of catalysis of catalases, peroxidases, and P-450)involve heme-mono-oxveenase enzvmes (cvtochrome ... . . the tbrmntion of oxidizing intermediates rhat are formally "I.'r(\'j"species. In moit cases thesrructure is an Ferl\'1=0 derrylj renrrr ctmhined umith a porphvrin r-cation radirsl (see r t v i t v s in reierenrr 7 for more e s t m d e d disrussion of these remarknI)k spt.cies1. The heme-m(8no-oxygenns1.s are important dt.toxifvina enzymes in the liver and arc tnvolved in drug metabnlism and, paradoxically, in the oxidative nrtivation of carcinogenic hydrocarbons. In oxidation of deuteroferriheme with H 9 0 9the "Fe(V)" intermediate is not observed because it is rapidly reduced by unoxidized deuteroferriheme t o a formal Fe(1V) intermediate which is probably a n Fe(II1)-porphyrin ;-cation radical complex.
--
Giher Important Effects of Heme Dimerfzation Simple ferrohemes (Fe(I1)-porphyrins) are rapidly oxidized by oxygen to ferrihemes (by reactions involving fioeroxo dimers). If this reaction was not inhibited hv the proteins, the reversible oxygen carriers hemoglobin and mvodohin could not work! Some fascinatine model svstems havebeen developed (by synthesis of ~ ~ i i k fence" et and "capped" porphyrins) in which the ferrohemes can reversibly bind 0 2 . For a good review see reference 8. Analytical Application of Chemiluminescence Since the clock time depends on the concentration of ascorbate (or other suitable reducing agent) the method has potential utility in analysis. Sensitivity can be increased by using photomultiplier detection of the endpoint rather than visual detection and in suitable cases the method can be made specific using enzyme techniques (9). Chemiluminescent methods of analysis are likely to become increasingly important because of their high intrinsic sensitivity. One important area is the development of chemiluminescent immunoassay methods which could replace assays involving radioactive materials.
Llterature Cited (1) Hasgett,M.L.:Jones. P.:Oldham,K. B. J . C h m . E d u c . 1963.40.387. (21 .Inner. P.:Hagget,, M.L.; Loo~idge..J.L.J. Chrm.lidur. 1964,41,610. (91 White. E. H. J. Chem.Educ. 1957.34.275. 141 For n rexiew pec:Jones. P ; Wllrm, I. In M e l d Inns i n B ~ R I U ~S~(rem: CYI Sigel. H.. Ed.: 1978. 7.186. (Sl Jones, P.:Manlle,D.;Wilson. I. J . Chem. Lzc., Dalton Trans. 1983,161. (61 .loner. P.: Scowen. N . , Photnchem. Phntahinl.. in preai. 171 Dunfnrd. H.R.:Oniphin,D.:Raymond,K. R.:Sieker,L.,Eds.ThaBialogiralChemism o l l r o n : NATO~ASISerieb C, Vol. 89: Reidol: 1982. (8) Syker. A. G . In Aduancer in Inorganir ond Rioinorponic Mechanisms: Sykes, A. G., Ed.:Acsdemic:New Yark, 1982: Vnl. 1.p 121. (91 ~ r e wJ.. E.:~oner.P. A n d L e t t 1985.1~.1579.
Volume 64
Number 1
January 1987
71
