| llsl n ‘1 A! I} {1' w u I m l! ,1 \ . I. 1 ‘ 1‘. ‘ ‘l ‘ l I -‘J—‘ 'i’E‘fEflMiHATSQN (”.125 3C 23-525 3‘5 OXli‘éfiflGN WlTH CESAR fi’ERCHLQRATE six??? AQfi-QRPEON OF CSARBGN 913231333; Thesis {m flit Dogma of M: S. MECE'RGAEQ $TA‘E‘E UNEVERSETY flami‘a $3 mfiezzugkas “196252 WHESIS (1.2—- w: L272}? FRY FF; f" {fr-'5‘. State \T " . ... ', . a, . ‘ ‘ 1" -3 "1""."EJ‘LI'Y . : W5 5 ’ 5 A ”3.543335 ”1-3.“: W" MTCHTGAN STATE UNIVERSITY or AcmrutwRE AND «me sumac EAST LANSING, MICHIGAN 0/531 4 7 0/: £sz DETERMINATIW OF KETOSES BY OXIDATION WITH CERIC PERCHLORATE AND ABSORPTIw OF CARBON DIOXIDE By Damte G. Sallmuskas AN ABSTRACT Submitted to the College of Applied Science of Michigan State University in partial fulfillment. of us requirements for the degree of MASTER OF SCIENCE Department. of Chemistry 1960 mnute G. Salkauskas 1 ABSTRACT A. method for determining ketoees has been developed which depends on the oxidation of these substances by ceric perchlorate followed by absorption of the evolved carbon dioxide on Ascarite. mmwmnw.. EXPERIMENTAL . . Apparatus . Materials . Original 0xidation.Procedure Determination of the Rate of Formic.Acid Oxidation Revised Oxidation Procedure . TABLE OF’CONTENTS D O RESULIS AND DISCUSSION . . . . . . e . SUMMARY 0 O O O C C O O O O O C O O O EmumRAPHY I O O D O O O I O O O O O C O O C O O C O O O Page 11 12 12 1h 18 19 INTRODUCTION lhe various eerie salts and their oxidizing properties have been known for a long time. Since 1860 occasional suggestions have been made for their use in analytical procedures. In 1861 Lange (1) recognized the oxidizing properties of ceric sul- fate and suggested its use as a volumetric reagent. Sonnenshein (2) in 1870 recommended it instead permanganate for the titration of iron. In 1899 A. Job (3) mentioned the stability and strong oxidizing properties of acid solutions of ceric salts. He noticed that there was possible a substitution of ceric salts for permanganate in cases where the latter is not applicable, as in the analysis of oxalchlorides. The use of ceric sulfate as a quantitative oxidizing agent was first suggested by Barbieri (u) in 1905. Barbieri described two pro- cedures for determination of nitrous acid. One was a direct titration of nitrite with ceric sulfate in which the end.point was determined.by s disappearance of the yellow color of ceric ion. In the second pro- eedure excess eerie sulfate was used and the unreduced eerie ion was determined iodometrically. Nitrite solution could contain nitrates and eerie sulfate other rare earths without causingany difficulty in these determinations. Barbieri also noted that hydrazine and hydroxyl- amdne reduce eerie compounds in cold solution. Sommer and Pincas (5) found that eerie sulfate as well as other eerie salts could be used for quantitative oxidation of hydrasoic acid In this work.Sommer and.Pincas treated hydrazoic acid in neutral or acidic solution with an excess of ceric salt and then measured the evolved nitrogen gas. The amount of nitrogen evolved corresponds to the reactions 20c“ + M3 - 3N,+2Ce”+2H" More recently nartin (6) used eerie sulfate for oxidation of hy— drazine. 'ihe reagent was added in excess and then back-titrated iodo— metrically. Martin reported that the precision obtained in this deter- mination lies within 0.1 per cent. Benrath and Ruland (7) tried the eerie oocidetion of various organic compounds, such as hydrazine, tartaric and mlic acids, anthraeem, hydroxylamine as well as thiosulfate, sulfurous acid and hypophosphorous acid. Someya (7) titrated eerie salt potentiometrically with steward ferrous sulfate, while VanName and Fenwiek (7) develOped graphs to de- scribe the potentiometric titration of ceric sulfate with titanous sul- fate. ' Application of eerie oxidinetry to determination of organic sub- stances has been studied by a number of investigators. Hillard and Young (8) applied oxidation by eerie ion to determination of organic acids, such as maleic and tartaric acids. Similarly, Mean and Wallace (9) determined hydroquinone by eerie oxidation, Chapin (10) p~amino~ phenol, and White (11) a variety of organic compctmds. Smith and his co-workers (12, 13) made a research into the theory of oxidizing action and developed better quantitative methods for oxi-- dation of a rumber of organic compounds including alcohols and carbo- hydrates. Shanna (1h) rechecked previous studies of carbol’wdretes and organic acids and developed better techniques for quantitative oxidations. 3 lhe rather late development of ceric oxidimetry can be attributed to early difficulties in determining the end-points of these reactions, and until the potentiometric method for an end point was developed, my extensive work in this field was impossible. Thus, most of the early titrations with ceric sulfate employed pokntiometric determinations of end points. However, in 1931 Walden, iiammet and Chapmn (7) found that ferrous-o-phenanthroline' complex ion, now known as retrain, could serve as a reversible oxidation~reduction indicator of high potential. In its reduced form the ferrous o-phenanthroline complex possesses a deep red color, «while the oxidized form is pale blue. The ferric complex was found to be resistant to acids and to action by permangamte, ceric or dichromte ions in acid solution. In 1931.; Hamet, Halden and mm (7) in their study of high potential indicators prepared derivatives of phenanthroline and diphenylamine. They found that nitrophemnthrolim ferrous ion, called nitroferroin, has a potential of 1.25 volts and is more stable to acids than ferroin, ahich possesses a potential of 1.11; volts. The discovery of these indicators mde possible siwile visual determination of end points in eerie mcidimtry. Some of the advantages of ceric sulfate and other ceric salts can be sumarised as follows! i. Ceric salts are easily prepared. 2. Solutions of lceric salts can be easily and accurately stand- ardieed against readily available prim standards under a wide variety of experimental conditions. 3. Solutions of an. salts are stable even on boiling (15). (Ceric perchlorate solution was found to be rather sensitive to light). h 1;. Solutions of ceric salts can be used in excess for oxidation in hot or cold solutions, then back-titrated in a sulfuric, perchloric or nitric acid medium with a standard reducing agent. 5. Carla ion has a rapid and positive oxidizing action. 6. Oxidations by ceric ion can be adapted to a number of differ- ent indicators. They can be followed also by potentiometric techniques. he use of nitrato and perchlorato cerate anions as a rapid qual- ' itative test for alcoholic hydroxyl group was developed by Duke and Smith (16). This test was based on the intense red color given by hexanitrato potassium or ammonium cerate upon solution in alcohols. The color suggested the possibility of the reaction of an alcohol with the complex nitrato cerate ion. The same type of results were obtained with hexaperchlorato cerate ions, except that the color produced in some cases was less permnent than with the nitrato cerate test solutions. Alcohols having up to ten carbon atoms, hydromrcarboxylic acids, and carbohydrates (such as glucose, sucrose and dextrins) gave positive tests. In the case of carbohydrates the color produced was rapidly lost as the result of oxidation. Hillard and Young (17), in their determination of hydroxy carboxylic acids with permanganate found that there is a slow and variable oxida- tion of formic acid, which was an intermediate product. Then they con- firmed Benrath and Ruland's observation that formic acid was not 03(- idized with ceric sulfate even on boiling. Therefore, theoretically, S in oxifition of tartaric acid by ceric sulfate six equivalents of oxygen would be required per molecule of tartaric acid if it is oxidized to formic acid only. If oxidation proceeds completely to carbon dioxide and water 10 equivalents of oxygen would be required. But Willard and Young found that, under conditions employed by them, the tartaric acid required 7.2 equivalents of oxygen per molecule. Similar observations were obtained with glycolic, mile and mlonic acids. No explanation was offered (for the results. Sharma (18) repeated Hillard and Young’s work and obtained similar results. Show attempted to explain his results in the following man- nor: I'Ii' the organic acid decomposed via formic acid only, then the above observations suggest that although formic acid is not oxidised appreciably when present alone, yet its oxidation is induced by the energy evolved in the oxidation of other organic molecules.... Another plausible explanation might be that a portion ofthe organic acids is not oxidized via formic acid, but by some other mechanism in which its oxidation proceeds completely to carbon dioxide and wh'aterdI In reinvestigating the oxidation of formic acid with ceric sulfate, Shanta found that a slight moxmt of reagent was always consumed by formic acid. Thus it seemed that ceric sulfate can oxidize the formic acid but at a much slower rate. An increase in acid concentration seemed to increase the oxidation of formic acid. However, oxidation seemed to be complete in 50 minutes in the presence of 66 per cent (by volume) of sulfuric acid in the reaction mixture. 6 Thus Sharma showed that formic acid can be oxidized quantitatively by ceric sulfate in the presence of concentrated sulfuric acid. Sharma also showed that, by increasing the concentration of sulfuric acid, maleic, fumaric, benzoic, phthalic and salicylic acids can be oxidized with ceric sulfate completely to carbon dioxide and water, while succin- ic and acetic acids are not oxidized at all. Sharma and.Mehrotra (18) confirmed Smith and Unke's (l9) observation that glycerol is oxidized to formic acid not only with ceric perchlorate but also with cerate sulfate. Glycerol is not oxidized to tartronic acid, as was believed by Cathill and.Atkins (20). But the carats formic acid oxidation still disturbed Sharma and his co-workers and.sgain they repeated the experiments with.pure ceric sulfate (21). Then they found that the oxidation was negligible. The impurity catalyzing the reac- tion in the other experiments was found to be chromium,'while iron, manganese, silver, osmium.and other rare-earth elements showed no ap~ preciable catalytic influence. The action of ceric perchlorate or ceric sulfate on reducing sugars was also studied. It was found that sugars containing aldehyde group are oxidized to formic acid only: or) (gen), + 6[o) : 6HCOOH z0H The ketcnic sugars also have formic acid as an end product, but in addi- tion, the ketonic group is oxidized to carbon dioxide. zon -o ( 03;, + 7[o1 --——---—> SHCOOH+COZ+H20 2 Smith and Duke (22) on treating glucose with ceric perchlorate found the oxidation to formic acid.was complete in hS minutes and.A. A. Forist and J. C. Speck, Jr. (23) by two other procedures found all sugars tested, with the exception of DL-glyceraldehyde, to be complete- ly oxidized.within one hour. Sharma (1h) studied the action of ceric sulfate on reducing sugars and obtained the following results. Observations with.Glucose , A; :fl'VOIume ‘O.lh5H Reflux- Ce(SO4)z Equiva- Glucose Glucose 1‘ of Ce(504)z ing consumed lent: of found present glucose added time oxygen solution consumed (m1.) (ml.) (min.) (m1.) (9.) (9.) 2 (S 5 2.h2 10.52 .0052? .00600 2 5 10 2.68 11.65 .00583 .00600 2 5 15 2.7h 11.97 .0059? .00600 2 5 30 2.73 11.91 .00595 .00600 2 5 60 2.7h 11.97 .0059? .00600 2 S 120 2.7% 11.97 .0059? .00600 3 S 10 3.81 11.05 .00829 .00900 3 S 15 h.03 11.73 .0087? .00900 3 5 20 £1.11 11.98 .0089; .00900 The above data show that glucose is oxidized to formic acid within _..____ 15 minutes if about 100 per cent excess of ceric sulfate is present. The time required for complete reaction increases if the excess of ceric ‘ sulfate is less. Observations with Fructose volume 0.1b5 H Reflux- Ce(504)z Equiva- Fructose Fructose of Ce(504); ing consumed lents of found present izgfitogz added time 4 cgfixgfigd (ml.) (ml.) (min.) (ml.) (9.) (9.) 2 5 10 3 .065 12.82 .00572 .00626 2 5 15 3.355 111.01. .00626 .00626 2 5 30 3.3h 13.97 .0062} .00626 2 S 60 3.3h 13.97 .00623 .00626 2 5 120 3.355 lb.0h .00626 .00626 1 S 10 1.666 13.9h .00311 .00313 1 S 15 1.676 111.02 .00313 .0031} The results in the above table show that 1h equivalents of oxygen are consumed.per mole of fructose oxidized. Carbon dioxide was detected as one of the products of the reaction. This ability to produce carbon dioxide from.the ketonic group on the oxidation with the cerate reagent has found an.applieation in tracer studies also. .H. Calvin and his coaworkers (2h) in their study of path of carbon in.photosynthesis used this method of oxidation of carbonyl carbon.of a ketose to carbon dioxide uhich.uas precipitated out and counted.as barium carbonate. In our study an attempt was made to develop a method.for a quanti~ tative determination of detoses present by measuring the amount of car- bon dioxide absorbed on.Ascarite. EXPERIMENTAL Apparatus: The apparatus for carbon dioxide absorption on semi-micro scale was set up as shown in Figure l. The vessels were connected with im» pregnated rubber tubing which was made in the following way: Rubber tubing of medium thickness was cut into pieces of 3-11 inches in length and placed in a suction flask containing melted wax. Then the wax was heated in a boiling water bath and the rubber placed in it for 30 min? utes, continuing the heating under reduced pressure until no more air bubbles could be seen coming out of the rubber tubing. This step was repeated several times . Materials. The following reagents were used in these experiments: 0.5 M Ceric perchlorate solution in 6 N perchloric acid (G. F. Smith Chemical Co. reagent) Ethylene glycol . 0.18 N and 0.2 H sodium oxalate in 0.1 M perchloric acid Nitro-ferroin indicator, 0.02514 0.28 H ceric perchlorate in h M perchloric acid 0.01 to 0.03 N perchlorate estate in 2 u perchloric acid Formic acid D—Fructose D—Sorbose Dim'drmqyacetone ll L-Arabinose Ascarite Thgnesium perchlorate (anhydrous) Concentrated sulfuric acid ‘ Nitrogen gas Original Oxidation Procedure. The apparatus was set up as shown in Figure l and the following oxidation procedure was carried out: About 0.5 millimole of fructose (or other sugar) was weighed accurately on an arelytical balance and placed in the reaction vessel. It was dissolved in approximately 1 ml. of water. Then the Ascarite-filled absorption tube was wiped with a damp 'cloth, dried with a clean, dry cheese cloth, allowed to equilibrate next to the analytical balance for 5 minutes and weighed. Afterwards it was connected back into the system. The stopcocks were opened to allow the nitrogen to pass through the system and the flow of nitrogen was adjusted to 5 ml. per minute as measured by the displacement of water. Then about 20 ml. of approx- imately 0.5M ceric perchlorate reagent was slowly introduced through the side arm of the vessel by the use of a syringe. The reaction was allowed to proceed for 3 hours. Then the gas flow was stopped, the Asmrite tube containing the absorbed carbon dioxide was cleaned in I the same manner as described above and weighed. The Ascarite tube was then reconnected and the system swept with nitrOQen for an additional 30 minutes. The weighing procedure was repeated. The additional sweep- ings and weighingswere made for several times, but the constant weight was never obtained. 12 The same procedure was repeated with L~arabinose and formic acid. Determination of the Rate of Formic Acid Oxidation. The procedure of Forist andSpeck (23) was followed with the follow- ing exceptions. Method I To 88 mg. of formic acid placed in a dark flask and kept at 30°, 50 ml. of approximately 0.2% eerie perchlorate reagent was added. Ten-ml. aliquots were removed at intervals during the oxidation and added to 15 ml. of 0.18}! sodium mlate. The excess of oxalate was titrated with 0.03}! ceric perchlorate reagent to a nitro-ferroin end point. - The same procedure was repeated for the oxidation at 20°C. Method II To 88 mg. of formic acid in a dark flask at 20°, 25 ml. of approx- imately 0.5M ceric perchlorate was added. One-ml. aliquots were removed from time to time and added to 3 ml. of 0.21! sodium oxalate. The ex- cess of oxalate was titrated with 0.03m ceric perchlorate to 9. nitro- ferroin end point. Revised Oxidation Procedure. Since the oxidation of fructose was found to be complete in 15 min— utes when it was performed in the same way (Method 1) as the formic acid oxidation at 20° (see Table III), the following oxidation procedure was deve loped: 13 To a reaction vessel kept at 20°C. a weighed amount of D~fructose was added and discolved in 1 ml. or water. The.Ascarite tube was cleaned and weighed as described previously. The system was connected together and the flow of nitrogen.adjusted to h—S ml. per minute as meaaured.hy the displacement of water. Then about 18 ml. of 0.5M ceric perchlorate reagent was slowly introduced into the reaction vessel and the reaction allowed to proceed for 15 minutes. At the end of this time 7-8 ml. of ethylene glycol was added to stop the reaction and the nitrOQen flow was adjusted to 7~8 ml. per minute. The sweeping was continued for 2.25 hours and the.Aacarite tube was cleaned and.weighed. .Additional aueepingsof 15 minutes were made and the tube was reweighed. If the increase of weight was not greater than 0.5 mg. the sweeping was con- sidered complete. 1.17;an zerbaaonm 20;».qu - . z, . J - . ..i 3.39; 55> owii mmak zoComomdq . a at .. ¥QWdI.UZOU IF.3 Gunfight Winwgmmbm Q.U< U.&DIln—Dm ’E-m..fl- . _\ ' -_/ 1' >1 ,1 ”.‘P-‘r'r-M— ' ' \ / K I RESULTS AN D DISCUSSION The results in the original oxidation procedure were always high, as can be seen in Table I. Attempts to get better results were trade by shortening sweeping periods, but still about 110—118 per cent of the theoretical quantity of carbon dioxide was obtained. Even with arabin- ose there was an increase in weight in the Ascarite~filled tube. The possibility of the increase due to moisture was discounted due to the pnesence of three tubes filled with concentrated sulfuric acid and a. U-tube filled with anhydrous magnesium perchlorate preceding the Ascarite? filled tube. Another possibility was the oxidation of the formic acid which is famed during the oxidation of the sugars. Therefore, the oxidation of formic acid was tested under these conditions: the results can be seen in Tables II and III. -_ Sugar TABLE I Amount of Sugar Reaction Origiral Oxidation Procedure Mount of Goa-Absorbed Oxidized Time Found, mg. Calculated, mg. mg. minutes D-Fructose 89.8 fi fi 3 26.7 21.9 90.0 3 23.5 . 22.0 911.8 3 211.6 23.2 90.2 2 1/2 26.9 22.1 90.1 2 1/2 25.1 22.0 L-Al‘flblnosc 67.1]. 2 1/2 7 05 00° 91.1 2 1/2 7.9 0.0 Formic acid 88.0 3 1.6 0.5M ceri: perchlorate used for oxidation. 15 TABLE II (bridation of Formic Acid by Method I Time Mg. of Formic Acid Oxidized at minutes 30° 20° 0 6.203 5.851 7.70 0.00 15 10.351 10.90; 12.65 1.95 30 12.651 1h.953 17.25 h.30 60 17.25; 20.70; 19.55 6.10 0.28M ceric perchlorate was used for oxidation 88 mg. of formic acid was used for oxidation. TABLE III Oxidation of Formic Acid by Method II may“, Hg. 01‘ Formic Acid Oxidized at 20° 0 0.00 5 . 0.751 0.50 10 1.50; 1.50 1 2.003 2.25 2° 3.001 3.50 25 3.50; 3.75 3° use; 1.50 1‘0 5.00: 5.50 50 6.00; 5.50 60 6.253 6.00 0.5M ceric perchlorate used for oxidation. 88 mg. of formic acid was oxidised. As can be seen from the results a considerable amount of formic acid is oxidized during a 1-hour period at room temperature, while only a limited oxidation occurs at 20°C. 16 Since complete oxidation of sugar can.be obtained in 15 minutes with a weaker solution of ceric perchlorate (Table IV), the addition of ethylene glycol was introduced in the revision of the original oxidation procedure. The purpose of ethylene glycol was to react with the ex~ cess of ceric perchlorate present. It was noticed that during the addi- tion of the eerie perchlorate reagent to the sugar, or on addition of ethylene glycol to the eerie perchlorate, the solution would turn dark red, and the color faded quickly with sugars while it lasted longer with ethylene glycol. This type of observation was made by Duke and Smith (15) also. Results of the revised oxidation.procedure are given in Table V. The low results of sorbose and dihylroxy acetone were due to poor samples. Probably the materials were hydrated. Similar low results ‘ were obtained with these substances by Forist and Spock’s (23) method also. TABLE IV Oxidation of Fructose at 20° by Method I fin” m M of Fructose Oxidized minutes 9. 1 75.27; 62.25; 67.50 15 96.05; 95.253 98.25 30 93.53} 97.503 99.75 60 101.10; 101.25, 102.75 90 mg. of Fructose was oxidized. .28M ceric perchlorate used for oxidation. 17 TABLE V Revised 0xidation.Procedure for Ketoses Amount 'TheoretioaIQYield“ .Actual Yield Sugar Oxidized calculated mg. Hg. mg. Fructose 90.6 22.2 22.h 90.3 22.1 6 91.6 22.11 22 h 91.6 22.h 22 7 90.8 22.2 21 3 90.0 22.0 21 8 90.8 22.2 19.3 93.2 22.8 19.8 Sorbose 92.2 22.5 17.7 9200 22.5 1708 Dinydroaqr 92.0 2.5 .0 37.6 acetone 90.3 hh.l 37.0 Constant temperature bath at 20° was employed. 0.5M.ceric perchlorate used in oxidation. Ethylene glycol was sdded.after 15 minutes of reaction. Total sweeping time was 2 l/2 hours. SWARY The history of the development of ceric oxidiaetry was traced. The determination or ketoses by the absorption or carbon dioxide was attempted and a method for ceric perchlorate oxidation was develop- ed. Fructose, sorbose and dihydroxy acdtom were oxidized by this method. The rate of formic acid oximtion was checked and it was found that there is considerable amount of oxidation at room temperature at high ceric perchlorate concentrations, while only slow oxidation occurred at 20°C. 10. 11. 12. 13. 1h. 15. 16. 17. 18. 19. 21. 22. 23. H. r. A. G. F. J. P. H. N. J. 1.. G. G. H. r. F. H. R. G. R. ii. G. A. J. B IBLICERAPHY H. Hillard and P. Young, J. Am. Chem. Soc., g9, 1322 (1928). L. Sonnershein, Ben, 2, 631 (1870). Job, Compt. rend., $29.: 101 (1899). Barbieri, China. Ztg., 32, 668 (1905). Sommer and H. Pincus, Ben, 113, 1963 (1915). Martin, J. Am. Chem.,Soc., 19;, 2133 (192?). Young, Anal. Chem, £11., 152 (1952). H. Hillard and P. Young, .1. Am. Chem. Soc., 2, 11.9 (1929). H. human and J. H. Wallace, J. Am. Chem. Soc., 53, lth (1930). w. Chapin, Master's Thesis, Michigan State University, 19M. White, Eater's Thesis, Michigan State University, 1939. F. Smith and c. A. Getz, Ind. Eng. Chem, m1. Ed., 19, 191 (1938). F. Smith and c. A. Gets, Ind. Eng. mom, Anni. 121., 12, 301. (1938). N. Sham, Ami. Chin. Rota, g, 1123 (1956). R. Duke and G. F. Smith, Ind. Eng. Chem, mi. 34., 33, 339 (19110). R. Duke and G. F. Smith, Ind. Eng. Chem, Anal. Ed., '13, 201 (19110). H. Willard and P. Young, .1. Am. Chem. Soc., 2, 132 (1930). 11. Shanna and R. c. Mehrotra, Anal. Chim. Acta, 13,, 1:17 (1951;). P. Smith and r. R. Mo, Ind. Eng. amt, Aral. Ed” 1;, 558 (19111). outhiu and c. Atkins, J. Soc. Chem. Ind. (London), 89 (1938). 11. Shaun Ind R. c. Mehrotra, Anal. Chim. Acta, 1;, 1.19 (1956). F. Smith and r. R. Duke, Ind. Eng. Chem, m1. 3.1., 15, 120 (191.3). A. Forist and J. c. Speck, Jr., m1. Chem, 31, 1166 (1955). A. Bassham, A. A. Benson, 1.. D. Kay, 11. 2. Harris, A. '1‘. Wilson and 11. Calvin, J. Am. Chem. Soc., 16_, 1760 (1951i). T V ”iii/)7” m Iii/I171 iii/7i" 11171111111)?in in?“ 1293 031