Because of the low solubility of high molecular weight branched paraffins and cycloparaffins in acetone, some modifications in technique are necessary for fractions above Ctl. Satisfactory results have been obtained up to C30by operating with a 75-25 v mixture of acetone and tetrahydrofurane.
ACKNOWLEDCMENT
The authors thank C. B. Willingham of the Mellon Institute for the loan of the thermal diffusion column and R. A. Brown of the Esso Research and Engineering Company for the mass spectral analysis of the branched paraffins.
CONCLUSION
Selective adsorption chromatography with Sephadex LH-20 provides a method for the separation of types of hydrocarbons for which other adsorbents are relatively ineffective, such as the separation of paraffins from cycloparaffins and alkylbenzenes from cyclanobenzenes.
RECEIVED for review February 15, 1967. Accepted March 23, 1967. Work supported by the American Petroleum Institute and the American Chemical Society Petroleum Research Fund Grant.
Spectrophotometric Determination of Micro Amounts of Iodine and Glucose with Fluorescein Donald E. Braunl a n d W. Hugh Wadman Unioersity of the PaciJic, Stockton, Calif.
THEUSE OF THE IODINATION of fluorescein as a means for the micro determination of iodine using visual colorimetry is described by Harlay ( I ) . The product formed in a neutral, buffered solution is the 4,5-diiodide of fluorescein. The application of this method to glucose determination depends on the formation of iodine from excess periodate remaining after the periodate oxidation of glucose. In a boric acid-sodium borate buffer (pH of 7), the iodide-periodate reaction is ( 2 ) : 104-
+ 2 I- + 2 H'
+
IP
+ HsO +
103-
(1)
The iodine is then reacted with fluorescein. EXPERIMENTAL
Apparatus. Absorption measurements were made on a Beckman D B spectrophotometer with recorder ; light path was 1 cm. Reagents. All solutions were made with deionized water. The disodium salt of fluorescein (Uranin) was used. Potassium periodate was used as the source of periodate ions. The iodine-potassium iodide solution was prepared with a 1 : 5 weight ratio. The buffer was prepared from 6.66 grams of boric acid and 0.32 gram of Na2B407.10H 2 0 in 200 ml of water. Sufficient buffer was added to all reaction mixtures to keep the pH near 7. Iodination of Fluorescein. Concentrations of iodine between 0.625 and 10.0 pg/ml of reaction mixture were reacted with buffered fluorescein solution in stoppered test tubes at room temperature. The concentration of fluorescein was 5 pg/ml for the lower concentrations of iodine and 10 pg/ml for the higher concentrations of iodine. A blank was similarly prepared without iodine. Absorption readings were made after 5 minutes. Iodide-Periodate Reaction. Concentrations of periodate ions between 0.8 and 8 pg/ml of reaction mixture were allowed Present address, Pacific College, Fresno, Calif. (1) V. Harlay, Aim. Pharm. Franc., 5 , 81 (1947). (2) E. Mulier and G . Wegelin, 2. Anal. Chrm., 52,755 (1913).
840
ANALYTICAL CHEMISTRY
10
0
2
6
4 12
8
10
(rs/ml)
Figure 1. Beer's law plots to react with 36.5 pg/ml of iodide ions in a buffered fluorescein solution. The concentration of fluorescein was the same as above. A blank was prepared without periodate. Studies were done a t 56" C (using a water bath) for a period of 10 minutes. Absorption readings were made after the tubes were cooled with water. Periodate Oxidation of Glucose. Glucose and periodate were carefully mixed with buffer in a glass-stoppered tube, the total volume being 5.2 ml. A reference was prepared without glucose. After the reaction was allowed to proceed for 20 minutes at 56" C , the tubes were cooled in water for 1 minute. Immediately, the glass stoppers were removed, and the stoppers and sides of the tubes were washed down with 2.5 ml of water. Next, 1 ml of iodide-fluorescein mixture and 1.3 ml of water were added, making the final volume 10 ml. A blank containing only buffer and iodide-fluorescein solution was also prepared. The tubes were shaken and heated for 10 minutes at 56" C , cooled, and the absorption readings were made. The concentrations per milliliter of reaction solution were: glucose, 0.04 to 2 pg; periodate ions, 7 p g ; iodidefluorescein, 36.5 pg iodide ions and 100 pg fluorescein.
GLUCOSE ( r s / m l )
Figure 2. Glucose determination Wavelength, mr A. B. C. D.
505 484
505 484
Concentration Determinations. The concentration of iodine was determined graphically (Figure 1). For glucose determinations, the differences between the maximum absorbances of the reference and the glucose solutions were plotted against glucose concentration on rectangular graph paper (Figure 2). RESULTS AND DISCUSSION
Absorption Spectra. The absorption peak of the iodinated fluorescein mixture occurs at 505 mk. When the cuvettes of reaction mixture and blank are reversed in the spectrophotometer, an absorption peak corresponding to fluorescein occurs a t 484 mk. Tht: absorption at both of these wavelengths is a measure of the amount of iodine reacting. Beer’s law is followed quite well at both 505 and 484 mp (Figure 1). Very slight deviations occur a t low concentrations and they are in accord with the literature (3). Similar results are obtained from the icldine solution and the iodine solution produced by the iodide-periodate reaction. The maximum absorption at 505 mp is affected very little in the pH range 6.4 to 7.8. Intense light evidently causes a photochemical degradation of the reaction product (4). Normal room lighting hiis virtually no effect up to 15 minutes. Thereafter, the absorptilm decreases slowly at room temperature. Virtually no effect is produced by the presence of 0.00125M Na+, 0.002M K+, 0.002,MI-, 0.09M and the following (3) S . Sheppard, Reu. Mod. Phys., 14, 312 (1942). (4)G . Karg, “Photodehalogenation of Fluorescein Dyes,” micro-
filmed doctoral dissertation, Polytechnic Institute of Brooklyn, N. Y . , 1963.
Temp, 98 98 56 56
O
C
Oxidation time, minutes 10 10 20 20
ions at a concentration of 0.003M: Cl-, HC03-, Mg+2,and Ca+2. Iodide-Periodate Reaction. Fluorescein was added before the iodide-periodate reaction was allowed to proceed so that the iodine produced would react immediately. This was particularly important at higher temperatures because of the ease of vaporization of the iodine. The reaction is almost instantaneous at room temperature in acid solutions, but is slower at higher pH’s. Buffered conditions were used because the subsequent iodination of fluorescein does not occur in acidic solutions. To hasten the reaction at the higher pH, a reaction time of 10 minutes at 56” C was used. Periodate Oxidation of Glucose. A temperature of 56” C was used for most determinations because the periodate oxidation of sugars is more easily controlled at moderate temperatures (5). The pH was kept near 7 because the following iodide-periodate reaction required a neutral solution. Some oxidations carried out at 98” C for 10 minutes yielded quite satisfactory results (Figure 2). The periodate-glucose molar ratio is 3.3 :1 for the highest concentrations of glucose determined. Because complete oxidation requires 5 moles of periodate per mole of glucose, complete oxidation of glucose does not occur under the conditions employed. Earlier studies have shown that the periodate oxidation of aldohexoses does proceed in steps (6, 7). Periodate is a nonspecific reagent and will oxidize 1,2glycols ; active methylenes; aminoalcohols; a-hydroxy ( 5 ) Y . Khouvine and G. Arragon, Bull. Soc. Chim. France, 8, 676 (1941). (6) S. Warsi and W. Whelan, Chem. Znd., (London) 1958, 71 (1958). (7) G. Lindstedt, Nature, 156,448 (1945). VOL 39, NO. 7,JUNE 1967
0
841
Table I. Iodine, rg/ml
Glucose, pg/ml
Precision Analysis CorreStandard sponding deviation, deviation, pg/ml trans.
4.0 7.94
zt0.4 f O .3 Zko. 3 f O .3 f0.4 ZkO. 3 f 0 ,3 f0.4
0.04 0.1 0.2 0.5 1 .o 1.5
Per cent deviation
zt0.05 f 0 .1 10.005 f0.004 f0.004 10.036 f0.034 zt0.036
1.3 1.3 12.5 4.0 2.0 7.2 3.4 2.3
aldehydes, acids, and ketones; and 1,2-diketones. Therefore, none of these compounds may be present in determining glucose by this method. The use of borate as a buffer may interfere with the periodate oxidation of sugars (8). Overoxidation (the consumption of more periodate by glucose oxidation than theoretically possible) is reported in the literature as a possible complication (6, 7, 9). However, analytical determinations are possible if the conditions used are kept constant. Determination of Jodine and Glucose. Typical transmittance-iodine concentration plots a t 505 and 484 mp are shown in Figure 1. (8) D. Hutson and H. Weigel, J . Chern. Soc., 1961, 1546. (9) G . Head, Nature, 165,236 (1950).
P!ots of the glucose concentration studies are shown in Cigure 2. The conditions were the same except for the differing temperature and time of the oxidation reaction. Two samples of known concentration are necessary to establish a reference line for each series of determinations. A precision analysis of typical iodine and glucose concentration-absorption studies a t 505 mp is shown in Table I. Four determinations were made for each concentration. Determinations a t 484 mp were less precise with the standard deviations being about three times greater for both iodine and glucose. The precision for the more complex glucose determination is the same as for the iodine determination. This method of iodine determination has extended Harlay’s method from a concentration range of 30 to 150 pg/ml of solution to between 4 and 10 pg/ml with good precision and accuracy. The application of this method for glucose determinations is useful for concentrations between 0.5 to 1.5 pg/ml of solution. Estimations are possible down to 0.1 pg/ml.
ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance of Russel Schmidt. RECEIVED for review November 14, 1966. Accepted March 16, 1967. This work was partially supported by a NSF Cooperative Graduate Fellowship and a NSF Summer Fellowship for Teaching Assistants.
Anion Exchange Separation and Spectrophotometric Determination of Titanium in Uranium-Plutonium Ternary Alloys Harold B. Evans and Rozetta R. Hallcock Argonne National Laboratory, Argonne, Ill. 60439
AN EXAMINATIONof the published literature on plutonium and plutonium alloys ( I , 2) covering the period through 1965 discloses only one published procedure dealing specifically with the analysis of titanium in plutonium nuclear alloys (3). Because titanium continues to find wide application in the nuclear field because of its beneficial metallurgical and nuclear properties, it was desirable to find new techniques which could be easily adapted to routine glove-box operations. The method proposed for the present application depends on the quantitative retention of plutonium and uranium on the resin column while the titanium passes through in the eluate where it is determined spectrophotometrically as the peroxy complex. The procedure now available (3) is based essentially on the precipitation of plutonium peroxide. The method is timeconsuming, has numerous centrifugation steps, and is plagued by gas evolution and the attendant controlled conditions E. A. Cernak, Pratt and Whitney Aircraft Division, MiddleConn.. CNLM-1802-3 (1959). .2; :6. i3. Evans and J . 0. Karttunen, CSAEC Repor! ANL-6956
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