edited by ROBERTREEVES Marlborough School 250 S. Rossmore Avenue LOSAngeles. CA 90004
Hemoglobinometry-A Biochemistry Experiment that utilizes the principles of ~rans-tion Metal Chemistry Vlncenzo Giullam La Salle University, Philadelphia, PA 19141 Jonn Paul Rleckl Vineland High School, Vineland, NJ 08360 Hemoglohinometry-the quantitative measurement o f , the concentration of hemoglohin in hlood-can serve as a unique, practical, and effective laboratory demonstration of the principles of transition metal chemistry. This experiment is well suited for second-vear hieh school chemistrv students, especially those gearei to the-health-care profelsions. For instructors desiring a multifunctional exercise, this experiment is particularly effective in that i t applies several chemical principles-transition metal chemistrv, colorimetry, and 6iochemistry-in a single exercise that is appealing to students. The porphyrins are unquestionahly the most important class of organic compounds-without them there could he no plant or animal life as we know it, since they play a key role in such important biological processes as photosynthesis and resniration. Hemoelohin. which is a coniueated . " nrotein containing an iron-complexed porphyrin known as heme, is the niement found in the bloodstream that is res~onsihlefor of oxygen from the lungs to the tissues, where the &&sport oxveen is consumed in the oxidation of such hioloeicallv .. i m i h t n n t suhstancesascarbohyrlmtes, lipids, and proteins. Hemoglobin is tdue in color, 11ut as it picks up oxygen in the lungs it is converted to bright red 6xyhemoglobin, which gradually gives up its oxygen to the cells and is reduced to hemoglobin again for its trip hack to the lungs ( I ) . The bond between hemoglohin and oxygen is a very weak one. and this chemical union in itself cannot he snoken of as oxidation, since the iron remains in the 2+ oxidation state when oxygen is bound to the hemoglobin molecule. The iron in hemoglobin, however, can he oxidized to the 3+ oxidation state, which will complex strongly with ions such as fluorine, cyanide, and hydroxide. Hemoglobin readily combines with cyanide ions, forming a dark red complex called cyanomethemoglohin. Because this complex is far more stahle than oxvhemo~lohindue to the ability of the cyanide ion to bind more tightly than oxygen to theiron in hemoglohin, oxygen will not replace the cyanide ions in the complex (2).I t is the remarkable stability of the cyanomethemoglohin complex that is exploited in this experiment. in hlood The auantitative measurement of hemoelohin " can be determined by colorimetric means (3).Since concentration is ~rooortionalto ahsor~tionaccordine to the BeerLambert the concentratfon of hemoglobin in a given samnle is determined hv first convertine it to the stable cyn~ornet.hemoglobincomplex and then comparing its ahsorption to the absorption of a known cvanomethemoelohin standard. The hlood samples used in this study were acquired with
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854
Journal of Chemical Education
special arrangements from a local hospital and veterinary clinic. If such facilities are unwilling to make available small quantities of blood for testing, studen- may choose to use their own blwd for study.The procedure involvessrertlizing an area of the skin with sterile gauze soaked with rubbing aI~.ohol.gently pricking the skin with a sterile hem ole^,^ and then squeering the hlwd into a small test tube. Ten drops of blood should suffice. Since hlood coagulates quickly, the t~loodsample should be tested immediately. Experimental Preparation of Cyanide Reagent In a 600-mL beaker equipped with a magnetic stirring bar place 1000 mg sodium bicarbonate, 50 mg potassium cyanide, and 204 mg potassium ferricyanide.Carefully pour 300 mL distilled water with constant stirring until the salts have dissolved. (Caution: KCN, although relatively harmless when handled with care, reacts violently with acids to evolve hydrogen cyanide gas, which is extremely toxic. As a protective measure, KCN should be handled under the hood with long rubber gloves to prevent skin exposure.) Carefully pour the solution into a l-L volumetric flask and fill to the l-L markine. - .(Caution: When not in use. the reaeent sh..uld he stored in a cod, dry place separate from uthcr rwrage and prc,tectad from acid*and oridmng materials.) Colorimetric measurements arp made with a spectrophotometer at 510 nm. The instrument is ralihrsted lor hemoglobin measuremmt hy fmt dctrrminmg the absurbanct*~ of various dilutions of a known cyanornethemoglobin standard5and then constructingaplot of concentration versus absorbance for the standard. Individual blood samples of 0.02 mL are prepared for colorimetric measurement by miring with 5.0mL of thecyanide reagent. Theconcentrain the sam~leis determined bv measurine the tion of hemoelobin " nhaorhnnce determinine the concentrationfrom the cancentra~ and ~ . . tion versus absorbance plot for cyanomethemoglohin standard. (Caution: As a protective measure, the cyanide reagent and blood samples should be drawn with rnicropipets. Long rubber gloves should be worn to prevent skin exposure.) ~~
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Author to whom correspondenceshould be addressed. Ref 1, pp 600-603, provides a good review of the basic principles of colorimetry and the Beer-Lamberl Law. The hemoiets may be purchased at some drug stores and at most surgical supply centers. A Bausch and Lomb Spectronic-20 visible light spectrophotometer was used in this laboratory project. The human cyanomethemoglobin standard used in this experiment (20 g/dL) may be obtained from Fischer Scientific,Chicago. iL 60607.
Table 1.
Adsorptlon and Concentratlon Data for Varlous Dllutlons of Human Cvanomethemodobln Adsorption
Table 2.
Concenhalion (g1dL)
Adsorptlon and Hernoglobln Concentration Data for Samples of Human Blood
Sample No.
.?ex (M or F)
Adsorbance
Concentration (g/dL)
1
F F F F M M F F M M F
0.328 0.338 0.345 0.353 0.426 0.447 0.356 0.314 0.402 0.429 0.335 0.337 0.453 0.435
13.0 13.5 13.8 14.1 17.0 17.9 14.2 12.6 16.0 17.2 13.4 15.0
2 3 4 5 6 7 8 9 10 11 12 13 14
F M M
18.1
17.4
Disposal and Waste Treatment All unused blood samples should be returned to the hospital or veterinary clinic where they were obtained. Unused cyanide reagent should be sent in a labeled. sealed canister to a localchemical waste treatment facilitv. If this is not oossible. the reaeent mav he disposed ofsnfrly hy stirring withanexressirfhouseh
