A modification of the dry ash method for the protein bound iodine content of plasma or serum has been devised. This method permits completion of the determination in one laboratory day and produces results that compare favorably with the original two-day method. Recovery experiments were satisfactory and indicate no loss of iodine in the drying, ashing, or colorimetric procedures in the method

Routh, J. I.

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 70 Annual Issue Article 37 1963 A One-Day Modification of the Dry Ash PBI Method J. I. Routh State University of Iowa Let us know how access to this document benefits you Copyright ©1963 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Routh, J. I. (1963) "A One-Day Modification of the Dry Ash PBI Method," Proceedings of the Iowa Academy of Science, 70(1), 178-184. Available at: https://scholarworks.uni.edu/pias/vol70/iss1/37 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. 178 IOWA ACADEMY OF SCIENCE [Vol. 70 Melting point determination with a carbon arc image furnace is unique in that there is no container problem. Since the energy of the furnace is concentrated in a small area the specimen forms its own container eliminating a common source of error. Literature Cited 1. Bulletin of A. D. Little, Inc., Performance Data Sheet on ADL-Strong Arc Imaging Furnace, Cambridge, Miss., 1959. 2. Null, M. R. and Lozier, W. W., Rev. Sci. Instr., vol. 29, p. 163., 1958. 3. Null, M. R. and Lozier, W. W., J Appl. Phys., vol. 29, p. 1605. 1958. 4. McMahon, W. R., "High Temperature Spectral Emissivity of Some Ceramic Oxides," M.S. Thesis, Iowa State University, Ames, Iowa. 1962. 5. Butler, C. P., Research and Development Technical Report-USNRDL-TR-483, San Francisco, Calif. 1960. 6. Butler, C. P. and Inn, E. C. Y., Thermodynamic and Transport Proper-ties of Gases, Liquids and Solids," McGraw-Hill Book Co., Inc., New York, pp. 377-390. 1959. 7. Butler, C. P. and Inn, E. C. Y., Research and Development Technical Report-USNRDL-Td-235, San Francisco, Calif. 1958. 8. Martin, s_. B. and Lai, W:, "Ignition by Thermal ~adiation," Proc. Fire Res. Conf., Natl. Acad. Sci. Puhl. No. 475, pp. 84-92. 1956. 9. Janes, M., Graphite-Based Materials for High Temperature Applications, Natl. Carbon Co., Cleveland, Ohio. 1958. A One-Day Modification of the Dry Ash PBI Method J. I. RouTH1 with the technical assistance of Donna Douglas Abstract. A modification of the dry ash method for the pro-tein bound iodine content of plasma or serum has been de-vised. This method permits completion of the determination in. one laboratory day and produces results that compare fav-orably with the original two-day method. Recovery experi-ments were satisfactory and indicate no loss of iodine in the drying, ashing, or colorimetric procedures in the method. A method commonly employed for the analysis of the protein bound iodine content of serum or plasma was devised by Barker, et al, in 1950. The procedure was developed in this university and in modified form has been used in our clinical protein bound iodine laboratory since its inception. The method essentially con-sists of the precipitation of the plasma or serum proteins, washing of the precipitate, drying in the presence of sodium carbonate, ashing, dissolving the ash in an acid solution and colorimetrically measuring the disappearance of the color of eerie ammonium sulfate in the presence of sodium arsenite. Although many modi-fications of the method have been proposed, the time consuming overnight drying of the precipitate in the presence of sodium 1 Dept. of Chemistry, State University of Iowa. 1Routh: A One-Day Modification of the Dry Ash PBI MethodPublished by UNI ScholarWorks, 196319631 MODIFICATION OF DRY ASH PBI METHOD 179 carbonate results in a method that requires two days for its com-pletion. The present paper is concerned with a study of the attempts to shorten this drying period to permit complete analysis in a work-ing day. Figu re 1. Intfra-red lamp drying device . Experimental: Over ten years ago experiments were devised to reduce the time required by the PBI method. The h1bes containing the pre-cipitated plasma proteins plus the sodium carbonate solution were placed in a heated sand bath in a vertical position. The drying time could be reduced to about three hours, but even with constant attention the contents of some of the tubes would boil out the top. Heating blocks at 100° C. also dried the tubes in 2Proceedings of the Iowa Academy of Science, Vol. 70 [1963], No. 1, Art. 37https://scholarworks.uni.edu/pias/vol70/iss1/37180 IOWA ACADEMY OF SCIENCE [Vol. 70 about 3 hours with occasional boiling over. Since a 3 hour period of drying plus the required 2112 hours in the muffle furnace left insufficient time for the precipitation, washing and colorimeter procedures, attempts were made to further shorten the drying period. In the Clinical Chemistry Laboratories of the Mayo Clinic infra-red lamps are mounted over the tubes which are placed in a rack in an inclined position with a stream of air directed down the center of the tube. A similar apparatus was constructed in our laboratory (Figure 1) and its use resulted in , a more rapid drying of the contents of the tubes. Several short-comings were observed in this type of equipment. It was very difficult to adjust the heights of the lamps to produce uniform heating without the contents of some tubes boiling over. Also, the tubes in the rack did not dry uniformly and it required rotation of the tube positions to achieve rapid drying of all tubes. To overcome the difficulties of this procedure we again used cast aluminum heating blocks at 100° C. and found that it required about 3 hours to dry 20 tubes without attention. A special rack was constructed that fit over the top of the drying block and directed a fine stream of air into the center of each vertically held tube (Figure 2). The temperature of the block was increased to 106° C. and employing aslow stream of air the contents of 20 tubes could be dried within 60 to 90 minutes. The air flow through holes drilled with a No. 50 drill was found most effective at 80 ft. per minute. Figure 2. Aeration rack. Method: The one-day method in use in our laboratory involves several modifications of the original procedure ( 1,2) and will be present-ed in detail. 3Routh: A One-Day Modification of the Dry Ash PBI MethodPublished by UNI ScholarWorks, 196319631 MODIFICATION OF DRY ASH 1-'J:H METHOD 11H Precipitation: Specimens of serum or plasma, 1.0 ml., in duplicate, are diluted with 7 ml. of distilled water using a 10 ml. serological pipette with a large orifice. The 1 ml. portions of zinc sulfate and sodium hydroxide solutions are then added to precipitate the proteins. The tu bes are covered with a piece of parafilm and the contents are thoroughly mixed by inversion. Washing: After centrifugation, the supematent fluid is discarded and the precipitate is washed once with 10 ml of water. Thin footed stirring rods are used to completely suspend the precipitate. Other methods recommend multiple washings to remove inor-ganic iodides that may be present in the serum or plasma. In an extensive study of the washing process in our laboratory we found that a single washing removed inorganic iodides and that further washings failed to remove contamination from organic iodides that precipitated with the proteins. At the completion of the washing process the tubes are centrifuged and the supernatent fluid is discarded. Figuro 3. Instruments used for PBI determination . From left to right are th e Th er-molyne muffle furnace, the dri-bath with aeration rack in place, the special dri-bath containing 30 holes fro Klett colorimeter tubes, the Kle tt colorimeter tubes, the Kl ett colorimeter and tim er. The muffle furnace, l06°C. Dri-Bath and 30 hole 39°C. Dri-Bath were kindly furnished by Thermolyne Corporation, Dubuque, Iowa. The aeration rack, furnace and Dri-Baths as w ell as a muffle furnace holding 80 tubes and a small air compressor are available from Thennolyne. Drying: The 1 ml. of sodium carbonate solution is added to the washed 4Proceedings of the Iowa Academy of Science, Vol. 70 [1963], No. 1, Art. 37https://scholarworks.uni.edu/pias/vol70/iss1/37182 IOWA ACADEMY OF SCIENCE [Vol. 70 precipitate, the contents are mixed thoroughly with a Vortex stirrer, and the tubes are dried for 60 to 90 minutes in the dry bath set at 106° C. with a stream of air directed into the tubes as shown in Figure 3. The shortest drying time may be achieved by bringing the contents of the tubes to a boil before starting the air flow and turning off the air 10 to 15 minutes before the tubes are completely dry. Some experience is required to establish a pro-cedure for drying the tubes in approximately 60 minutes. Ashing: The dry h1bes are transferred to a muffle furnace which has been preheated to 600° C., and are ashed for 2112 hours at that temperature. The preheating of the muffle furnace saves time and is conveniently carried out by turning on the furnace when the tubes are started in the drying process. Colorimetry: The tubes are removed from the muffle furnace, cooled, and the HC1-I-LS04 solution is added to dissolve the ash. This so~ution is a mixture of HCl and H2S0 4 ( 23 ml. cone. HCl and 56 ml. cone. H 2S0 4 per liter) that contains in a 7 ml. volume the same amount of acid that was contained in the two 2 ml. aliquots in the original method. To the series of tubes containing the ash add first a stirring rod and then 3 ml. of the acid mixture. The rods prevent the acid solution from bubbling out of the tubes. When bubhling ceases add another 4 ml. of the mixture to· each tube in the series. Undissolved particles of carbon are separated by centrifugation and 3 ml. aliquots of the supernatent fluid are added to tubes containing 1 ml. of distilled H 20 and 0.5 ml. of the sodium arsenite solution. A maximum of 24 tubes per batch (Klett colorimeter tubes) are placed in a special dry bath, Figure 3, containing 30 holes and maintained at 39° C. The extra six holes are used to preheat tubes containing the eerie ammonium sulfate solution. vVhen the contents of the tubes have reached 39 ° C. ( 5 to 10 minutes), eerie ammonium sulfate solution is added to the series of tubes at 30 second intervals. Twelve min-utes after the addition to the first tube the colors are read in a Klett colorimeter at 30 second intervals. If more than 24 tubes have been ashed, this process may be repeated until all the colors are read. Results: Since our laboratory currently carries out approximately 100 FBI determinations a week, we have had ample opportunity to compare the results of the one-day method with those obtained by our earlier modification of the procedure. Essentially, the "old" two-day method referred to in Table I consisted of precipitation and washing procedures carried out on the first day with over-5Routh: A One-Day Modification of the Dry Ash PBI MethodPublished by UNI ScholarWorks, 196319631 MODIFICATIOJ\' OF DRY ASH PBI METHOD 183 Table I Comparison of typical results obtained by the one-day versus the two-day PBI method. Control Serum 1" Control Serum 2" Patients' Serum" 2 day 1 day 2 day 1 day Specimen 2 day 1 day µg/100 ml µg/100 mlu.g/100 ml µg/100 ml µg/100 ml µg/100 ml 4.7 5.0 10.7 10.6 A 3.6 4.0 5.0 5.2 9.3 9.7 B 6.4 6.1 4.7 5.0 10.5 10.6 c 4.0 4.2 5.1 5.0 10.2 9.7 D 5.0 5.4 4.7 5.2 10.6 10.6 E 4.7 4.7 4.4 4.7 9.7 9.7 F 1.3 1.3 4.4 4.4 10.3 10.2 G 10.2 10.2 4.7 4.7 10.1 10.2 H 2.3 2.6 "Range 4.5-5.1 "Range 9.4-10.6 "Patients from Thyroid Clinic µg/100 ml. µg/100 ml night drying of the precipitates in a forced draft oven. Ashing and the colorimetric procedures were carried out on the second day with a water bath at 39°C. employed in colorimetry. Typical results taken from our files are shown in Table I to illustrate the similar results obtained by both methods. Reco¥ery experi-ments in the original method of Barker, et al, ( 2) consisted of the addition of known amounts of iodine in the form of sodium iodide thyroxin or thyroglobulin to tubes containing precipitated and washed plasma proteins. To check the present method through the drying, ashing, and colorimetric phases, a known amount of iodine in the form of sodium iodide was added to the sodium carbonate solution prior to the drying process. Table II presents typical results of these recovery experiments. Table II Recovery experiments obtained from the one-day PBI method. Serum Iodine Total Value Added Calc'd. Found Recovery µg/100 ml ,ug/ 100 ml µg/100 ml µg/100 ml % ... 3.2 4.1 7.3 7.7 105.6 2.9 4.1 7.0 7.2 102.9 Specimen A B c ..... 8.9 4.1 13.0 13.2. 101.6 Patients D from E Thyroid F Clinic G Control Serum H I J Range 4.7-5.l Control .......... Serum .... Range 9.4-10.6 Discussion: 7.0 4.1 11.l 11.4 102.7 4.7 4.1 8.8 9.1 103.5 6.2 4.1 10.3 10.6 102.9 .. 18.7 4.1 22.8 22.1 97.0 5.4 4.1 9.5 9.4 99.0 . . . 5.0 4.1 9.1 9.0 98.9 6.5 4.1 10.6 10.5 99.0 4.7 4.1 8.8 8.9 101.2 5.0 4.1 9.1 9.3 102.2 4.7 4.1 8.8 9.1 103.5 .. ... 10.6 4.1 14.7 14.8 100.7 .. 10.6 4.1 14.7 15.3 104.0 ... 11.4 4.1 15.5 15.8 102.0 The protein bound iodine content of several thousand plasma and serum specimens has been determined in our laboratory 6Proceedings of the Iowa Academy of Science, Vol. 70 [1963], No. 1, Art. 37https://scholarworks.uni.edu/pias/vol70/iss1/37184 IOWA ACADEMY OF SCIENCE [Vol. 70 since the publication of the dry ash method ( 1,2). For several years the results of each batch have been controlled by the simultaneous determination of standard control sera. This con-tinuous experience has enable an adequate comparison of modifi-cations resulting in the one-day method. The typical results pre-sented in Table I point out the similar values obtained by both methods using control sera with different PBI levels and patients sera. The recovery experiments in Table II were designed to check every procedure in the method that would retain added inorganic iodide. It can be seen from these experiments that iodine was not lost in the more rapid drying process at 106° C. Literature Cited Barker, S. B. and Humphrey, M. J. 1950. Clinical determination of pro-tein-bound iodine in plasma. J. Clin. Endocrin. 10: 1136. Barker, S. B., Humphrey, M. J., and Soley, M. H. 1951. The clinical de~ermination of protein-bound iodine. J. Clin. Invest. 30:55. Sulfonation of Bathophenanthroline and Bathocuproine · RICHARD L. CRYBERG AND HARVEY DIEHL 1 4,7-Diphenyl-l, 10-phenanthroline, introduced in 1952 as a spectrophotometric reagent for iron by Smith, McCurdy and Diehl ( 1 ) , has proved useful and popular; some twenty papers ( 2) have now appeared detailing its application to the deter-mination of iron in sea water, wine, serum, urine, various metals, and other materials. This reagent was given the common name Bathophenanthroline. The great sensitivity of the reagent is made peculiarly useful by virtue of the solubility of its red, ferrous derivative in isoamyl alcohol, for extraction of the red compound from water into the immiscible solvent provides a concentration method making possible the determination of iron in concentra-tions as low as 0.002 parts per million, and even more im-portant, provides a method of removing the iron contaminants from the various reagents used in the analysis. The one disadvantage bathophenanthroline suffers is the occasional appearance of a turbidity when the analysis is per-formed entirely in the acqueous solution, especially when perchlorate is present. The English chemist Trinder ( 3) solved this problem by sulfonating bathophenanthroline by treatment with chlorosulfonic acid, the sulfonated product being com-pletely water soluble, even in the presence of perchlorate. Later Blair and Diehl ( 4) isolated and characterized the .sulfon.ated 1 Dept. of Chemistry, Iowa State University. 7Routh: A One-Day Modification of the Dry Ash PBI MethodPublished by UNI ScholarWorks, 1963

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A One-Day Modification of the Dry Ash PBI Method

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