Nd(CO0H)s 250 X mag-
Figure 3. nification
habits h a w been seen. These include needlcs, groups of nerdler, crystals showing pentagonal faces, a n d finally spherulites. X-ray data ( 2 ) indicate that all of these hanits-Le., needles, groups of needles, and spheres-are structurally the same, These data are also in agreement with those of Pabst (6, 7 ) and Xayer et al. (6)which indicate that the formates are hexagonal. Cryatals showing pentagonal faces degenerated into the habits shon-n in Figures 1 and 2 as crystallization
continued. Most of these photographs were taken by retarding evaporation of the test drop to isolate the form of interest. Sensitivity. Tests for t h e sensitivity of the method were made with lanthanum, t h e least soluble formate. A drop containing 10 pg. of lanthanum gives a characteristic precipitate in 5 to 10 minutes. Since precipitation with 50 pg. of lanthanum occurs immediately, 10 pg. seems t o be a reasonable lower limit. Interfering Ions. There do not appear t o be a n y other metallic ions which form spherulitic formates. Thorium forms a n insoluble formate b u t t h e crystal habit is cubic. Other cations investigated include the alkalis, alkaline earths, cobalt,, copper, uranium, and the SHIOH elements. LITERATURE CITED
(1) Chamot, E. AT., Mason, C. W., “Handbook of Chemical Microscopy,” Vo1. 2, p. 308, Wiley, Yew York, 1940. ( 2 ) Croft, W. J., Leipziger, F. D., Sperry
Rand Research Center, Sudbury, Mass., unpublished data, 1963.
Figure 4. Nd(COOH)3 250 X magnification, between crossed polarizers
(3) Haushofer, K., “Mikroskopische Reactionen,” p. 46, Friedrich Viewig, Braunschweig, Germany, 1885. (4) Leipziger, F. D., M.8.thesis, University of Massachusetts, Amherst, Mass., 1951. (5) Mayer, I., Steinberg, M., Feigenblatt, F., Glasner, *4.,J . Phys. Chem. 6 6 , 1737 (1962). (6) Pabst, A , , Am. J . Sei. 2 6 , 72 (1933). (7) Pabst, A., J . Chem. Phys. 11, 145 (1943).
(8) Sahoo, B., Panda, S., Patnaik, D., J . Ind. Chem. Soc. 37, S o . 9 (1960). RECEIVEDfor review August 8, 1963. Accepted September 23, 1963.
Constant Current Potentiometric Titration of Ascorbic Acid and Ascorbic Acid-Glutathione Mixtures C. 0. HUBER and H. E. STAPELFELDT’ University of Wisconsh - Milwaukee, Milwaukee, Wis.
b Various methods have been developed to estimate ascorbic acid, using 2,6 dichlorophlsnol indophenol, N-bromosuccinimide, cind various other oxidizing reagents. These methods involve visual, potentiImetric, and amperometric end poini detection. The proposed constant current potentiometric method requires simple apparatus and provides relatively rapid, convenient, and sensitive determination of ascorbic acid. Samples of 15 to 450 pg. per ml. ‘were successfully titrated at the 1 to 2% error level. Titration curves are presented and interpreted. A procedure for titration of ascorbic acid and glutathione in the same solution is presented and discussed.
-
A
-
of chemical methods for determining ascorbic acid have been developed. Most of them are slight variations of the classical U.S.P. method (4) involving the dye, 2,6dichlorophenol-indophenol, as the standard oxidant. E d y modification NUMBLR
of this method t o include potentiometric end point detection was developed by Harris, Mapson, and T a n g ( 3 ) . This method required a special mercuryplatinum “double-electrode” in addition to the saturated calomel electrode. Although good results were obtained for the samples tested, a fresh doubleelectrode had to be prepared frequently. I n a n effort t o remove this necessity, Spaeth, Baptist, and Roberts (6) developed a variation requiring a simple platinum electrode as the working electrode. This method is somewhat simplified, b u t t h e potential change at the end point is rather small and difficult t o detect in dilute concentrations. In addition, t h e results obtained are dependent on the previous history of the platinum electrode. Apparently platinum oxide surface layers are involved in the activity of their electrodes. Barakat, El-Wahab, and El-Sadr ( I ) developed a method of assay using X-bromcsuccinimide as the standard oxidant in a n iodide system, resulting in a n iodonietric titration n i t h visual starch end point. Good re-
sults were obtained in the concentrations studied. Coulson, Crowell, and Friess (2) determined ascorbic acid and total ascorbic acid and glutathione amperometrically by iodometric titration n i t h standard iodate. This procedure involved two separate titrations. In one sample the ascorbic acid was oxidized by a n excess of 2,6-dichlorophenol-indophenol, then the glutathione was oxidized with standard iodate. A second sample was titrated with standard iodate to the total ascorbic acid-gluthathione end point. B y comparison of t h e two titrations, the concentration of ascorbic acid was determined. Constant current potentiometry has not previously been applied t o the titration of ascorbic acid or of glutathione. This work involved the development of accurate, convenient, and relatively rapid procedures using this technique. Titrations using 2,6dichlorophenol-indophenol, S-bromoPresent address, Department of Chernistry, Purdue University, Lafayette, Ind. VOL. 36, NO. 2, FEBRUARY 1964
315
respectively. Ascorbic acid aliquots containing between 0.5 and 12.5 mg. were withdrawn from the sample solutions and diluted to approximately 75 ml. in order to cover the electrodes. The titration mixture was stirred as the titrant was added. Titrant increments were smaller near the end point, of the order of 0.10 ml. or less. Potential readings were taken 20 seconds after each addition of titrant, noting the direction of potential drift. The end point was marked by a lower potential that was stable for at least 20 seconds. Figure 1.
Voltammetric data for 2,6-
dichlorophenol-indophenol vs. ascorbic acid titration 1. Oxidolion of ascorbic acid 2. Oxidation of reduced 2,6-dichlorophenolindophenol 3. Cathodic reriduol current 4. Reduction of excess 2,6-dichlorophenolindophenol
succinimide, and iodate as standard oxidants were developed. Titration curves are interpreted in terms of voltammetry. Some comparative aspects of the methods and the simultaneous determination of ascorbic acid and glutathione in the presence of each other are presented. EXPERIMENTAL
Apparatus. Current-voltage curves were prepared using a manual polarograph. The saturated calomel electrode was connected t o t h e sample container by a salt bridge having sintered glass ends a n d containing 0.1N acetic acid. The constant current apparatus used was similar t o t h a t described by Reilley, Cooke, and Furman (5). Electrode pairs consisted of gold foil (4-sq. cm. area) connected to gold leads, or flattened gold wire (1-sq. em. area). Titrations were carried out in a low-form 150-ml. beaker. Solutions were stirred with a magnetic stirrer during the titration. A commercial p H meter with a 0- t o 1400-mv. potential scale was used to follow potential differences between the electrodes. Ordinary volumetric burets were used. Reagents. Both the ascorbic acid and glutathione were reagent grade ch,emicals. The 2,6-dichlorophenolindophenol was Fisher's No. 5-286 and the E-bromosuccinimide was Eastman's KO. P-5922. Other chemicals used were reagent grade. All chemicals were used as received. Procedure. 2,6 - Dichlorophenolindophenol and Nrbromosuccinimide solutions were prepared by dissolving t h e appropriate portions in hot water (followed by filtering in t h e case of 2,6-dichlorophenol-indophenol), cooling, and diluting with distilled water. Samples were standardized against standard ascorbic acid solutions. Ascorbic acid samples were prepared in metaphosphoric-acetic acid (4) or acetic acid solutions of p H 2.0 and p H 3.0,
316
ANALYTICAL CHEMISTRY
Development and Description of Method. Various electrode materials
were investigated in order t o determine suitability for the constant current method. Current-voltage curves were considered for each of the following electrodes in phosphoricacetic acid solutions of p H 2.0: Au, Pt, graphite, Ag, Cu, Au amalgam, and Cu amalgam. The latter four materials showed large slopes for both cathodic and anodic currents, and on this basis were eliminated from further consideration. Gold, graphite, and platinum gave approximately similar anodic waves for oxidation of reduced 2,6-dichlorophenol-indophenol and for oxidation of ascorbic acid. Cathodic currents are due to reduction of dissolved oxygen. Further currentvoltage curves were obtained to determine the extent of the cathodic potential shift due to unreduced 2,6-dichlorophenol-indophenol. It was determined that graphite and gold undergo sizable potential shifts and are suitable electrode materials. Gold was chosen because of ease of electrode fabrication. The titration of ascorbic acid with 2,6-dichlorophenol-indophenol can be interpreted in terms of the currentvoltage curves taken at a gold electrode shown in Figure 1. When two electrodes are polarized by a constant current, the potential of each electrode is established by the intersection of the 10-pa. constant current line and the currentvoltage curve. Prior to the addition of any titrant solution, the potential difference is indicated by points A and C. With the first addition of titrant the anodic wave shifts from curve 1 (oxidation of ascorbic acid) to curve 2 (osidation of reduced 2,6-dichlorophenolindophenol) and the measured potential difference drops. The potential difference then remains essentially constant until the presence of excess 2,6dichlorophenol-indophenol shifts the cathodic wave from curve 3 to curve 4 and the characteristic end point potential drop is noted. The potential difference read after the end point corresponds to points B and D. A typical titration curve is shown in Figure 2. The constant current potentiometric titration of ascorbic acid with standardized A'-bromosuccinimide can be de-
R
400
P Y
300'
7 (1 .
0 0
2
4
6
8 1 0 1 2 1 4
LL OF DYE
Figure 2. Titration curve for 2,6-dichlorophenol-indophenol vs. ascorbic acid
scribed in much the same way. Figure 3 shows current-voltage curves obtained at a gold electrode for this titration system. Initially, before the addition of any oxidant, the potential is indicated by points Ill and -V. .Is the titration proceeds the ascorbic acid concentration becomes depleted, and the anodic wave approaches curve 1. At the equivalence point neither ascorbic acid nor -Y-bromosuccinimide is present in solution, and the relatively large potential difference measured is indicated by points L and S. Once past the end point, excess X-bromosuccinimide causes a cathodic potential shift from curve 3 to curve 4 and the potential is indicated by points I, and P. If in addition to ascorbic acid the sample solution contains iodide, slightly different results are obtained. The anodic wave is held a t curve 5 and the potential is indicated by points Q and N prior to the end point (Figure 3). The A'-bromosuccinimide added oxidizes iodide, which in turn preferentially oxidizes ascorbic acid. Once past the end point, excess free iodine is present and the cathodic ivave shifts from curve 3 to curve 5, resulting in a potential drop. Typical titration curves for the two systems involving .V-bromosuccinimide and X-bromosuccinimideiodide are shown in Figure 4. When standard iodate v a s used as the oxidant for the ascorbic acid-iodide system similar results were obtained. A A simultaneous determination of ascorbic acid and glutathione by double titration in the same solution was developed. The sample mixture \\as titrated with 2.6-dichlorophenol-indophenol solution to the first potential drop corresponding to the ascorbic arid end point. This was follolved by the titration of glutathione with iodate in a n iodide medium, until the second potential drop was noted. Errors as a result of slow reaction between 2,6dichlorophenol-indophenol dye and glutathione are discussed below.
4
5
i
.
3
Figure 3. Voltarnrnetric data for N-brornosuccinirnide-ascorbic acid titration 1.
2. 3. 4. 5.
Anodic residuol current Oxidation of arcorbis: acid Cathodic residuol current Reduction of excem N-bromosuccinimide Iodide-iodine current.voltage plot
RESULTS AND DISCUSSION
Investigation of reproducibility of titration results are summarized in Table I, for both :2,8dichlorophenolindophenol and X-bromosuccinimide as titrants. Data for various concentrations of prepared saniples as well as for some fruit juices are yiven. Experimental results with weighed amounts of reagent grade ascorbic acid are presented in Talde 11, which also includes comparisons of end points determined electrochemically using N bromosuccinimide alone and with iodide, visual and electrochemical end points, and experimental results for some practical sample assays. Titrations of ascorbic acid and glutathione in common solution by the method outlined aboi e were performed. Three titrations of solutions containing 0.859 mg. of ascorbic acid and 1.207 mg. of glutathione consistently yielded a 2% positivc relative error for ascorbic acid and a 2 to 5y0 negative relative error for glutathione. Discussion. Results obtained experimentally showed good reproducibility for both preptred and natural samples of ascorbic acid. Titration solution concentrations of from 15 t o 450 pg. per ml. were used, but end points were unmistakable even a t the lower limits. Althoug? not investigated here, it seems certEin that smaller amounts than the lo ,ver limits stated could be titrated successfully using titration cells of smaller volume. Concentrations of ascorbic acid ranged from 10-6X I O 10-251 in the final titration mixtur.. Good results were obtained for 2,6-dichlorophenolindophenol titrant concentrations of 10-4M to 10-*M. The lower limit of concentration for the N-bromosuccinimide titrant was 10-3M. Below this
concentration, the oxidant decomposes too rapidly. Both the 2,6-dichlorophenol-indophenol and N-bromosuccinimide visual end points showed some positive error at lower concentrations (see Table 11). Agreement between electrochemical and visual end points was good at titrant concentrations of l O - 3 M and greater. Titrations can be carried out in less than 10 minutes. The apparatus and procedure used to obtain experimental results were simple and convenient for use in both colored and clear samples. Prior to the end point, slow drifting was observed with each addition of titrant. The measured potential difference first dropped, then recovered to a higher value. This drift was attributed to slowness of the reaction between ascorbic acid and the standard oxidant. Each addition of oxidant caused a temporary excess which was removed by slow reaction with the ascorbic acid still present. Unstable potential readings observed by other workers (3, 6) before the end point can also be attributed to this cause. For the ascorbic acid-indophenol titration curve (Figure 2), a slight rounding was noted in the region of the end point. This rounding was attributed to the irreversibility of the current-voltage curve for the reduction of 2,6-dichlorophenol-indophenol.The first addition of titrant resulting in a lorn-er potential reading with no upward drift was taken as the true end point. No such rounding was noted for the N-bromosuccinimide or N-bromosuccinimide with iodide methods, because of the more reversible waves for the oxidant. I n these cases the end points were taken to be the peak in the titration curve and the first potential drop respectively. I n the ascorbic acid us. 2,6-dichlorophenol-indophenol system a cathode film formed during the titration. This film appeared similar to an apparent condensation product of reduced 2,6dichlorophenol-indophenol which precipitates in the titration solution after the end point. It is thought that a film
Table
II.
Sample soln. Ascorbic acid (reag. grade) 98.8 pg. /ml. 18.9 pg./ml. Vitamin C tablet Orange juice Ascorbic acid (tech.)
A
'"""1 800.
600.
A
1
400-
Y
200-
i
7 ;
;
;
s
1
WL. N-BROMOSUCCINIMIDE
Figure 4. Titration curve for N-brornosuccinimide-ascorbic acid 1.
2.
Without KI KI present
of this condensate accumulates on the cathode because of electrolytic reduction of 2,8-dichlorophenol-indophenol a t the cathode. Successive titrations in a series resulted in slightly increased measured potential dif-
Table 1. Reproducibility of Results Using 2,6-Dichlorophenol-lndophenol and N-Brornosuccinirnideas Standard Oxidants
Sample soh. 7.1 x 1 0 - 4 ~ ascorbic acid Cranberry cocktaila 9.98 x 1 0 - 4 ~ ascorbic acidb Orange juice0
Av. concn. rg./ detns. ml. No. of
Std. dev. PP./
ml.
5
125.0
0.2
3
105.3
0.1
4 174.7 0.2 5 217.0 2.0 a 2,G-Dichlorophenol-indophenol as std. oxidant. b hi-Bromosuccinimide as std. oxidant.
Results for Various Titration Methods
2,G-Dichlorophenol-indophenol, X-Bromosuccinimide, wg./ml. dml. 99.1,99.4 19.2,19.4 206,221, 217, 224 331,38i1, 332
Identical to preceding sample but XI added KI added Identical to preceding sample but end point detected visually using starch
97.A.97.9 --.i9.2,19.5 242,226,229 348,340,345 447,452,447 451;444' - 2
174, 174, 173, 174 176, 177
VOL. 36, NO. 2, FEBRUARY 1 9 6 4
317
ferences, but these increases did not interfere with the potential drop at the end point. Beyond the end point the potential slowly drifted to smaller values. When the potential of each electrode was observed separately vs. a nonpolarized reference electrode, i t was ascertained that the drifting occurred only at the cathode. The drifting is probably attributable to increased effective surface area at the cathode due t o accumulation of the condensation film. Various current levels were used in conjunction with both electrode materials in an attempt to sharpen the end point for the 2,6-dichlorophenol-indophenol method. Gold electrodes gave best results for current density values between 2 and 10 pa. per sq. cm. Be-
low 2 pa. per sq. cm. the potential was established by the residual current, while above 10 pa. per sq. cm. the electrodes were less sensitive to excess 2,6-dichlorophenol-indophenol. The determination of ascorbic acid in common solution with glutathione proposed by Coulson, Crowell, and Friess ( 2 ) is based on the assumption that the reaction rate for the reaction between 2s6 - dichlorophenol and glutathione is negligible. The results reported above show consistent positive error for the indophenol-ascorbic acid end point, but negative errors for the glutathione-iodate end point. This indicates slow reaction between the 2,6-dichlorophenol-indophenol and glutathione before the first end point for ascorbic acid is reached. Because
of this slow reaction, the simultaneous titration method, although rapid and convenient, is limited to the several per cent error level. LITERATURE CITED
z., El-Wahab, M. F., El-Sadr, RZ. hi., AKAL. CHEM. 27, 536 (1955). Coulson, D. Crowell, IT. R.7 Friess, S. L., Zbzd., 22, 525 (1950). (3) Harris, L. J., Mapson, L. W,, \Tang, Y. L., Biochenz. J . 36, 183 (1942). (4) "Pharmacopoeia of the United States of America," 16th rev., P. 66, black Printing Co., Easton, Pa., 1960. ( 5 ) Reillev, C. N,, Co&, w, D., Furman, N. H., ANAL.CHEM.23, 1223 (1951). (6) Spaeth, E. E., Baptist, V. H., Roberts, Martin, Ibzd., 34, 1342 (1962). RECEIVEDfor review jUly 24, 1963. Accepted October 17, 1963. (1) Barakat, hi.
Ultratrace Analysis of Metals with a Curved Crystal X-Ray Milliprobe C. L. LUKE Bell Telephone laboratories, Inc., Murray Hill,
b A new x-ray fluorescence method for the ultratrace analysis of metals has been developed. The metal to b e determined is first isolated by conventional chemical methods. It is then taken up from water or dilute acid solution by a tiny disk of a strongacid type cation exchange resin or a strong-base type anion exchange resin and determined b y x-ray analysis using a fully-focused curved-crystal x-ray milliprobe. The method can b e used for the trace analysis of 28 or more cationic metals or 15 or more anionic metals. As little as 0.01 pg. of the more sensitive metals can b e determined.
T
HE SENSITIVITY of the borax diskx-ray method for the trace analysis of metals ( 2 ) is not adequate for the analysis of many high purity materials used in solid-state research and development. I t appeared desirable, therefore, to try to extend the sensitivity by taking advantage of the inherent high sensitivity of a curved-crystal x-ray milliprobe. I n order to use this instrument efficiently the sample to be analyzed must be obtained uniformly distributed in a low atomic number matrix of small area and volume. This can be accomplished by taking up the isolated trace metal to be determined in a small disk of a cation or anion exchange resin sheet (1). This approach has been thoroughly
318
ANALYTICAL CHEMISTRY
N. 1.
explored and has proved to be very successful. It has been shown that up to 20 pg. of 28 or more cationic metals can be taken up in a cation exchange disk and determined. As little as 0.01 pg. of the more sensitive metals can be separated and determined. This sensitivity is two orders of magnitude better than that possible with the borax disk method. I n like manner it has been shown that trace amounts of 15 or more metals or elements can be taken up in an anion exchange disk and determined. EXPERIMENTAL
Apparatus and Reagents. The xray instrument used was a General Electric spectrometer converted into a fully-focusing curved-crystal x-ray milliprobe. It employed an FS 7 5 tungsten target tube and was operable in the region of 20" to 95" (28); or 35" to 95" (28) when a He path was used. The size of the spot of exciting x-rays striking the sample was controlled by a l / ~ inch diameter gold aperture. A LiF crystal and a N o . 6 (xenon filled proportional) counter was used in the analysis of all metals whose fluorescent radiation is shorter than that of TiKg. For T i and longer wavelength metals, a n EDT crystal with a No. '7 (flow proportional) counter was used. I n practice, the instrument was operated at maximum power-Le., 7 5 ma. and 50 k.v.p.-using a He path wherever possible. The K a lines were used hherever possible. The x-ray crystal was adjusted to give maxirnum counts
with the particular metal being analyzed. The punch used t o cut out the small resin disks was made from hardened steel and was designed to cut out a disk lis inch in diameter. This diameter \\as sufficiently great so that the spot of exciting x-radiation could be fully contained within the circumference of the disk. The punch was provided with a spring-controlled ejector to release the cut d i k Since the ion exchange resins shrink on drying, it was necessary, to obtain disks whose area was uniform, to cut the disks while the resin was completely dry or completely wet. I n the present a o r k the disks were cut from wet resin sheet. The support for the disks, wed in the x-ray analysis consisted of a sheet of 0.25-mil Mylar film supported between two concentric phenol fiber rings. The Mylar film was smeared uniformly with a thin film of petroleum jelly and the dried resin disks were pressed lightly on the l l y l a r film so that they were lying flat. Precautions were taken to ensure that the distance between the bottom of the gold aperture of the milliprobe and the top of the disks was reproducible and as small as possible. The strong-acid type C-60 cation exchange sheet resin and the strongbase type A-60-2 anion exchange sheet resin supplied by American Machine and Foundry Co. of Springdale, Conn., were used. They \yere cleansed of contaminating metals and the anion exchange resin was converted to the OH or C1 form in the usual manner (3). They were then washed relatively
