1594
Anal. Chem. 1984, 56. 1594-1600
preliminary evaluation of the method indicates that it is suitable for the quantitation of triazolam over a similar concentration range. ACKNOWLEDGMENT The design and conduct of the clinical phase of this study by M. Kramer, M. Scharf, T. Joyce, R. E. Pyke, R. N. Straw, and W. Veldkamp are gratefully acknowledged. We thank M. Dopheide for preparation of the manuscript.
Registry No. AIprazolam, 28981-97-7. LITERATURE CITED Hester, J. B.; Rudzik, A. D.;Kamdar, B. V. J . Med. Chem. 1971, 14, 1078- io8 I. Hsl, R. S. P. J. Labelled Compd. 1973, 9 , 435-442. Eberts, F. S.,Jr.; Philopoulos, Y.; Relneke, L. M.; Vliek, R. W. Pharmacologist 1980, 22, 279. Sethy, V. H.; Harris, D. W. J. Pharm. Pharmacol. 1982, 34, 115-116. Eberts, F. S.. Jr., The Upjohn Company, unpublished work, Adams, W. J. Anal. Lett. 1979, 72, 657-671.
(7) Greenblatt, D. J.; Divoll, M.; Moschitto, L. J.; Shader, R. I. J. Chromatog.. 1981, 225, 202-207. (8) QII, M.; Kamdar, B. V.; Collins, R. J. J. Med. Chem. 1978, 21,
1290-1294. (9) Beyer, W. F.; Gleason, D. D. J . Pharm. Sci. 1975, 64, 1557-1560. (IO) Fenimore, D. L.; Davis, c. M.; Whltford, J. H.; Harrlngton, C. A. Anal. Chem. 1978, 48, 2289-2290. (1 1) Bombardt, P. A.; Brewer, J. E.; Adams, w. J. "Book of Abstracts", 185th National Meeting of the American Chemical Society, Seattle, WA, March 24, 1983;American Chemical Society: Washington, DC,
1983; 183. (12) Karger, B.; Giese, R. Anal. Chem. 1978, 5 0 , 1048A-1073A. (13) Smith, R. B.; Gwllt, P. R.; Wrlght, C. E. Clin. Pharm. 1983, 2 , 139-143. (14) Gibaldi, M.; Perrier, D. "Pharmacokinetics", 2nd ed.; Marcel Dekker: New York, 1982,Chapter 2. (15) Metzler, C. M.; Elfrlng, G. L.; McEwen, A. J. Blometrlcs 1974, 30, 562-563.
RECEIVED for review December 20, 1983. Accepted April 12, 1984. The work reported in this paper was presented in part a t the 33rd APS National Meeting, APhA Academy of Pharmaceutical Sciences, San Diego, CA, 1982.
Liquid Chromatographic Separation and Polarographic Determination of Aqueous Polythionates and Thiosulfate Bokuichiro Takano,' Michael A. McKibben,2and Hubert L. Barnes* T h e Pennsylvania S t a t e University, Ore Deposits Research Section, University Park, Pennsylvania 16802
Anlon exchange Separation of polythionates and thlosulfate by HPLC followed by dlfferentlai pulse polarographic determlnatlon provides a systematlc and sensitive analytical method for mlxtures of these aqueous sulfur species. The time necessary for the partial separatlon of thiosulfate and polythlonates Is 12 mln. Polarographic determination of each ion can be performed wlthin 5-15 mln except for the tlme necessary for sulfltolysls (20 mln) wkh an error of less than f10% for lo-' to loW3M of thiosulfate and polythionates.
Polythionates are commonly formed as intermediate oxidation products of sulfide minerals at acidic to neutral pH (I, 2). Such oxidation is primarily responsible for acid pollution of rivers and lakes receiving drainage from mining activities. However, understanding of the reaction mechanisms of this process has been impeded by the inability to analytically resolve the intermediate sulfur species thus formed, especially the polythionates. The many published papers on analytical methods for polythionate solutions (3) are deficient to various degrees in (1)ability to separate each thionate, (2) sensitivity, and (3) simplicity and rapidity of the procedures. Published methods have been based mainly on cyanolysis or sulfitolysis of thionates to form thiocyanate and/or thiosulfate ions which are measured spectrophotometrically (4-9). In order to determine each species, the authors proposed rather complicated procedures involving, for example, temperature or pH controls, catalytic reaction, and reaction time control. An alternative basis, the electrochemical behavior of POlythionates, has been described by several authors (10-19), Present address: The University of Tokyo, College of Arts and Sciences, Kornaba, Meguro-ku, Tokyo 153, Japan. Present address: Department of Earth Sciences, University of California, Riverside, CA 92521.
However, the polarographic analysis of polythionates is difficult because of their overlapping half-wave potentials (3). Recent developments in high-performance liquid chromatography (HPLC) have provided a means for rapid separation of mixtures of polythionates (20-22), but the methods still have some problems including a low sensitivity to trithionate. We have developed a simple and comparatively sensitive method for mixed polythionates in aqueous solutions, based on HPLC separation plus polarographic determination.
EXPERIMENTAL SECTION Reagents. I. Polythionates. Potassium trithionate was prepared following the method by Stamm et al. (23). Potassium tetrathionate was obtained by recrystallization of commercially available reagent (BDH Chemicals). The method described by Goehring and Feldmann (24) was used for the preparation of potassium penta- and hexathionates. Purity of these reagents was checked by IR spectra (25) and chemical analysis (6). The tri-, tetra-, and pentathimates were obtained as pure salts, and the hexathionate salt assayed 97.5%. Polythionate stock solutions M) were prepared by dissolving these reagents in distilled water except for trithionate solutionwhich was prepared as needed because it degraded at the rate of 5% per day (This rate is slower than that expected from the results of Naito et al. (26).) II. HPLC Mobile Phase. Sodium citrate (Fisher Certified Reagent) was purified once through recrystallization. The mobile phase (pH 5.0) was prepared by dissolving 294 mg of sodium citrate and 112 mg of citric acid into a liter of triply distilled water; this solution, loT3M in sodium citrate, was deaerated by boiling before use. III. Reagents for Sulfitolysis. A pH 3.5 buffer solution was prepared by mixing 80 mL of 0.5 M acetic acid with 5 mL of 0.5 M sodium acetate solution. A carbonate buffer solution of pH 9.9 was also mixed by adding 50 mL of 0.3 M sodium bicarbonate solution to 50 mL of 0.3 M sodium carbonate solution (6). Sodium bisulfite (0.15 M) and formaldehyde (0.5 M) solutions were prepared by dissolving Fischer Certified Reagent Grade Chemicals. IV. Supporting Electrolytes for Polarography. Both 2 M cesium chloride solution and 1M tetramethylammoniumchloride (TMC) were made by dissolving Sargent Welch and Baker
0003-2700/84/0356-1594$01.50/00 1984 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984 3
2R
L
0
2
4
6
8
1
0
1
2
1
4
Retention time (minute)
Flgure 1. Separation of thiosulfate and tri-, tetra-, penta-, and hexathionates by high-performance liquid chromatography: column, Applied Science SAX; mobile phase, M sodium citrate aqueous solution (pH 5.0) at 1.0 mL m i d ; detector, UV at 218 nm; sensitivity: 0.5 AFU; sample size, 100 ML sample, 2 X10-3 M thlosulfate and polythionates. The peaks are (1) thiosulfate, (2) tri- plus tetrathionate, (3) pentathionate, and (4) hexathionate.
Reagents, respectively. The solutions were separated from particulates with an 0.45-pm Milipore filter. A phosphate buffer (pH 6.5) was prepared by mixing equal volumes of 2 M potassium dihydrogen phosphate and 2 M disodium phosphate. These reagents were recrystallized once before use. Another phosphate buffer (pH 5.0) was made by mixing 10.30 mL of 0.2 M disodium phosphate with 9.70 mL of 0.1 M citric acid. Liquid Chromatography. An Altex Model 100 high-pressure pump and an anion exchange column (Applied Science SAX, 250 mm long X 4.6 mm i.d.; 5-pm particle size) with a presaturation and a guard column (Applied Science) were used to separate thiosulfate and polythionates. All chromatogramswere obtained isocratically using citrate buffer solution at a flow rate of 1.0 mL/min. Sample solutions were injected by using 20- or 1 0 0 - ~ L sampling loops which were calibrated colorimetrically with ophenanthroline and a standard solution of ferrous ammonium sulfate. Less than 10 pequiv of sample anions was eluted. The separation of thiosulfate and polythionates was checked by a UV detector (LDC/Milton Roy Spectro Monitor I11 Model 1204D) at 218 nm. The eluents containing these anions were fractionally collected (2 mL each), diluted to 4 mL, and analyzed polarographically. Figure 1 shows a typical chromatogram, indicating separation into three groups: (1)thiosulfate, (2) tri- plus tetraplus pentathionate, and (3) hexathionate. Polarography. Instrumental conditions were as follows. A polarograph (EG&G Princeton Applied Research Model 174A) with a static mercury-drop electrode assembly (Model 303) and an X-Y recorder (Houston Omnigraphic Model RE 0074) were used for polarographic determination of both thiosulfate and polythionates. The operating parameters were as follows 0.5 s drop time, 5 mV/s scan rate, 50 mV modulation amplitude, positive display direction, negative scanning direction, 3 V range, 1or 2 pA sensitivity, and differential pulse operation mode. In order to prevent oxygen from interfering with determinations of thiosulfate and tri- and tetrathionate, the mercury-dropassembly was enclosed in a small Plexiglasglovebox which was purged with nitrogen. The nitrogen used for deaerating the sample solution was purified by passing it through a heated (500 "C) Vycor tube filled with copper chips and through three scrubbing bottles, two of which contained 0.068 M ammonium metavanadate in 1.2 M hydrochloric acid over amalgamated zinc (27)and the other a 50% (v/v) ethanol-salt solution. I. Determination of Thiosulfate. The chromatographic eluent containing thiosulfate was analyzed by the standard addition method without adding any supporting electrolyte. Before analysis, samples were deaerated at least 4 min. Thiosulfate gave a well-defined polarogram which showed a half-wave potential of -0.16 V (vs. Ag/AgCl) at pH 5.0 (all half-wavepotentials given are for differential pulse peaks). 11. Determination of Polythionates. Two procedures were used, depending on the relative proportions of penta- and hexathionates as follows. Ila. Samples with Negligible Penta- and Hexathionates (less than 10% of other thionates). Following determination of the chromatographicthiosulfate eluent as described above, add 1mL of 2 M cesium chloride and 1 mL of TMC to 2 mL of the chromatographic eluent containing tri- and tetrathionates (Figure 1).
1595
Adjust the pH of the solution to 8.5 by adding 0.02 M sodium hydroxide solution. Deaerate the solution with nitrogen for 4 rnin and add 4 mL of distilled ethanol. Deaearate for an additional 8 min. Take a polarogram by scanning from -03 to -1.7 V (VS. Ag/AgCl). Tri- and tetrathionates showed reduction peaks at -1.42 and -1.16 V, respectively. Ilb. Samples with Penta- and Hexathionates Exceeding 10% of Other Thionates. They require a more complicatedprocedure. (i) Prior to HPLC separation, a determination of total sulfur present in thiosulfate and tetra-, penta-, and hexathionate is made by sullitolysisof a sample aliquot and polarographic determination of the resulting total thiosulfate, according to the reaction S,O$
-I-(X
- 3)S03'-
=
(X
- 3)S,02-
+ SsO2-
(1)
where x = 4 , 5 , and 6. (ii) Additionally, a determination of total sulfur present in thionates is made by sulfitolysis of a second sample aliquot and polarographicdetermination of the resulting trithionate, according to reaction 1given above. Following HPLC separation, separate determinations are made of the eluents containing (iii) thiosulfate, as described in section I, (iv) pentathionate, and (v) hexathionate. Analytical proceduresfor (i), (ii), (iii), (iv),and (v) are as follows: (i) Total amount of sulfur present in thiosulfate and tetra-, penta-, and hexathionates. Prior to HPLC elution, place less than 8 mL of sample solution containing to lo-* M thionates in a 10-mL volumetric flask. Add 200 pL of the carbonate buffer (pH 9.9) and 400 fiL of 0.15 M sodium bisulfite to the sample solution. Wait 20 min to allow sulfitolysis of tetra-, penta-, and hexathionates to thiosulfate and trithionate. Then add 300 pL of acetate buffer (pH 3.5) and 400 pL of 0.5 M formaldehyde solution,and allow to react for 5 min for completion of the masking of excess sulfite in the reaction mixture. Fill the flask up to 10 mL with distilled water. Take a polarogram by scanning from +0.1 to -0.5 V (vs. AglAgCl). The peak potential of thiosulfate in this supporting electrolyte is -0.18 V. Make a calibration curve using a standard solution with the same reagents for sulfitolysis. (ii) Total sulfur present in thionates. Take a separate sample solution containing more than 10" M total thionates. Add 200 gL of carbonate buffer (pH 9.9) and 400 pL of 0.15 M sodium sulfite to the solution. Wait 20 min and add 400 pL of phosphate buffer (pH 5 ) and 1 mL of 2 M cesium chloride. Deaerate the mixture with nitrogen for 4 min, add 3 mL of distilled ethanol, and again deaerate for 8 min. Take a polarogram of the solution by scanning from -0.9 to -1.6 V. The half-wave potential for trithionate is -1.31 V (vs. AglAgC1). Total trithionate concentration is determined by a standard addition method (cf. ref 27). (iii) See procedure for thiosulfate in section I. (iv) Pentathionate. Add 1 mL of phosphate buffer (pH 6.5) to the HPLC fraction containingtri-, tetra-, and pentathionates. Deaerate the solution with nitrogen for 4 min and take a polarogram with a scan from -0.3 to -0.8 V (vs. Ag/AgCl). The half-wave potential for pentathionate is -0.50 V. Make a standard addition analysis. (VI Hexathionate is determined by the same procedure as for pentathionate, using the same buffer solution and the HPLC fraction containinghexathionate. Make a polarographicscan from -0.3 to -0.8 V. The reduction wave for hexathionate appears at -0.39 V; this peak shifts slightly, depending on hexathionate concentration. In samples containing appreciable penta- and hexathionates, the procedures described above yield (i) total thiosulfate plus tetra-, penta-, and hexathionates as equivalent thiosulfate, (ii) total thionate as equivalent trithionate, (iii) thiosulfate, (iv) pentathionate, and (v) hexathionate. From reaction 1,it can be easily seen that tetrathionate and trithionate are calculated as: (vi) tetrathionate = (i) - (iii) - 2X(iv) - 3X(v); (vii) trithionate = (ii) - (iv) - (v) - (vi).
RESULTS AND DISCUSSION Stabilities of Penta- a n d Hexathionates in Citrate Buffer. As pointed out by Zezula (In,pentathionate solutions are unstable at any pH higher than 9. Stabilities of pentathionate solutions a t various pH values less than 7 were checked polarographically. Figure 2 shows that penta- and hexathionate anions are stable from pH 1.6 to 7 for at least
l5W
ANALYTICAL CHEMISTRY, VOL. 56. NO. 9, AUQUST iQa4
12
t
I
1 -4 at
B V
n
"
i I
fiequiv) which enables collection of a sufficient amount of the anions to be determined polarographically;and a weakly acidic mobile phase which prevents rapid degradation of polythionates during chromatographic separation (Figure 2). Incomplete HPLC separation of tri-, tetra-, and pentathionate anions was overcome by sulfitolysis together with polarographic analysis as described above. When a large size sample (for example, 5 pequiv) is injected into the column using a pH 4.5 citrate buffer solution, it exhibits a fairly complicated chromatogram. This may be caused by the following proposed reaction within the column (29):
+
i
L 0
M
40
60
80
1W
1x)
140
Time (nun)
Figure 2. Polarographic check of the stabillty of penta- and hexathionates in M citrate buffer: S,06*-, concentratlon, lo-' M; (A) pH 3.8, (B) pH 6.8, (C) pH 1.6; S60,*-, concentration, 2 X 10" M; (D)
pH 5.3,(E) pH 3.0.
Table I. Retention Times for Thiosulfate and Polythionate Anions (min)
column: Applied Science SAX, particle size 5 km, 250 mm X 4.6 mm i.d. flow rate: 1.0 mL/min sample size: 100 NLof 2.0 X M S2032plus polythionate solution
1h after preparation of the citrate buffer solutions. Therefore, the pH values of the HPLC mobile phase and the analytes for penta- and hexathionates were set at 5.0 and 6.5, respectively. Liquid Chromatography. The HPLC response to the mixture of thiosulfate and tri-, tetra-, penta-, and hexathionates is shown in Figure 1which indicates a satisfactory separation of thiosulfate and hexathionate ions from a group of tri-, tetra-, and pentathionate ions. However, a 60-cm column could not separate the trithionate-tetrathionatepentathionate group, except when a very small amount of these ions was charged to this column. Decreasing the sample size would yield unfavorable detection limits. No simple relationship was found between retention time and the sulfur-chain size of polythionate anions (Table I), although the general elution sequence occurs in the order of increasing size. Using a strong anion exchange column (Permaphase AAX, Du Pont) and an aqueous sodium citrate solution as a mobile phase, Wolkoff and Larose (28) obtained a linear relationship between the number of sulfur atoms of a thionate anion and the logarithm of its retention time. Explanation of the differences between their chromatogram and ours involves many factors. The difference in structure in the column-packing between Adsorbosphere SAX (Applied Science; totally porous and used in this study) and Permaphase AAX (superficially porous) is probably the main factor affecting the differences in chromatographic separations of the polythionates. Although complete polythionate separation was not fully realized in our procedure, the column we used has some advantages: short retention time (12 min) due to the short column length; large loading capacity (about 15
S,06'- + S203*+ H+= Sx+1062- HS03- (2) In order to separate the sulfur anions through the HPLC column without appreciable interactions among them, a mobile phase with M sodium citrate, pH 5.0, was used for the separation. Even when a 5-pequiv sample of thiosulfate plus polythionates was loaded into the column, the chromatogram showed good separation. If the pH of the mobile phase is set higher than 5.0, adequate separation of these sulfur compounds is not attained. Thus, setting the pH of the mobile phase to 5.0 is an important factor for stability and separation of the sulfur species. Polarography of Thiosulfate a n d Polythionates. Thiosulfate. In our analytical scheme for the sulfur compounds, thiosulfate is determined twice: first for the concentration of the sample and second as a sulfitolysis product of polythionates. As pointed out by Kolthoff and Miller (30) and Noel (31),the half-wave potential of thiosulfate anion becomes slightly more negative with increasing thiosulfate concentration. This was also observed in our samples. Noel (31) obtained a linear relationship between current and concentration for thiosulfate from 5 X lo-? to 1 X M in the differential pulse mode, while Renard et al. (32) reported a narrower range of linearity for thiosulfate in AC polarography. The calibration curve in this study was reasonably linear up to M. In the trace determination of thiosulfate, deaeration of dissolved oxygen must be carefully ensured because the oxygen reduction wave interferes with the thiosulfate peak. The detection limit for thiosulfate anion by DP mode polarography is as low as lo-' M, about the same as Noel's (31) results. Thiosulfate analysis was also used for the indirect determination of tetrathionate. The total amounts of tetra-, penta-, and hexathionates were measured as thiosulfate, which was produced through sulfitolysis of these polythionatesaccording to reaction 1 (6,33). Excess sulfite is masked by formation of a complex with formaldehydewhich does not interfere with thiosulfate at the dropping-mercury electrode (IO). A polarogram of the thiosulfate produced through sulfitolysis is shown in Figure 3. Tri- and Tetrathionates. As noted by Szekeres (3), polarographic analyses of polythionate mixtures has not been successful, because most members of this series have very similar half-wave potentials. Trithionate shows a half-wave potential more negative than other polythionates (34),but nontheless yields a poorly defined broad, overlapping peak with uneven current due to the other sulfur species. This is the major reason for the HPLC separation of polythionates prior to polarographic analysis. Tri- and tetrathionates are the major polythionate ions in soils (9),in lake sediments (35),in mining efluents (36),and in pyrite-leaching solutions (2). Therefore, solutions of these two anions should be more frequently encountered than those containing significant quantities of higher polythionates. Consequently, much effort has been directed in our laboratory toward developing a convenient analytical procedure specifically for the tri- and tetrathionates.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1984
1597
Table 11. Separation of Differential Pulse Polarographic Peak Potentials of Tri- and Tetrathionates supporting electrolyte
(aq so1n:alcohol E l:l, V/V)
1 M KC1-EtOHb 0.1 M KI-EtOH 1 M KI-EtOH 0.1 M NaC1-EtOH 0.5 M CsCl-EtOH 1 M LiC1-EtOH 0.5 M CsC1-0.1 M TMCC-EtOH 1 M NaC1-MeOHd 1 M KC1-MeOH 0.5 M CsCl-MeOH 0.5 M csc1-0.2 M TMC-EtOH 1 M CsC1-MeOH 0.5 M CsC1-0.3 M TMC-EtOH
s40-:
peak potential? V S3062- difference
-1.00 -1.12
-1.14 -1.27
0.140
-1.00
-1.15 -1.67 -1.33 -1.70 -1.40 -1.47 -1.37 -1.30 -1.42 -1.27 -1.43
0.150 0.160 0.170
-1.51 -1.16 -1.23 -1.18 -1.23 -1.12
-1.04 -1.16 -1.00
-1.15
insufficient peak separation
0.150
0.250
too close to Li current rise insufficient peak separation too close to Na current rise good separation but difficult to deaerate
0.260 0.260 0.270 0.280
good for determination good but difficult to deaerate good separation but depressed wave
0.190
0.220 0.240
Potentials vs. Ag/AgCl. *Ethanol. Tetramethylammonium chloride. Methanol. Several problems were encountered. The solutions previously recommended as the best supporting electrolytes were phosphate or tartarate buffers, but they did not work well in microanalysis of these anions because the maximum and minimum current suppressors which were used by several authors (10-12,37)were not effective in yielding a well-defined reduction wave for each anion. Alternatively, Subrahmanya (38) suggested water-ethanol (1:l)solutions of 1 M KCl or KI as a supporting electrolyte for determination of the lower (tri- plus tetra-) thionates. The DP mode polarographyof this mixture, however, exhibited two peaks whose half-wave potential difference was as small as 150 mV. This difference is inadequate to separate these peaks for quantitative determination of each anion. We also tested various aqueous salt-alcohol solutions for the simultaneous determination of tri- and tetrathionates, because some gave sharp and almost symmetrical polarographic peaks even at the microconcentration level of the anions and also, as shown by Naito et al. (26),hydrolysis of polythionates is suppressed by alcohol. When alkali halides were added as supporting electrolytes, the half-wave potentials of tri- and tetrathionates shifted toward less negative values in passing from lithium to cesium salts (17). As the peak potential of trithionate in these media is close to the final current rise of alkali metal ions, a larger cation is favored as a supporting electrolyte in order to shift the peak potential toward less negative values. Table I1 summarizes our resulta obtained by using different electrolytes for the quantitative polarographic analysis of tri- and tetrathionates. The ethanol-CsC1-TMC mixture is the best supporting electrolyte for separation of both thionate waves, because cesium ions sharpen tetrathionate peaks and shift both peaks toward less negative potentials. Furthermore, because ethanol dissolves less oxygen than methanol, the ethanol solution allows easier elimination of oxygen, whose wave just overlaps with the tetrathionate wave. As shown in Table 11, higher TMC concentration gives better separation of both peaks. However, too much TMC suppresses the waves and lowers reproducibility. The solution [0.5 M CsC1-0.2 M TMCI-ethanol (l:l,v/v) has been found to be the best compromise for good separation, sensitivity, and cost for the analysis of tri- and tetrathionates (Figure 3). Multivalent cationic salts were not included in this table, because their citrates are relatively insoluble in these media. The linear calibration curves for trithionate are shown in Figure 4,which also illustrates the variation in wave height of tetrathionate with increasing trithionate concentration. Tetrathionate up to 10 ppm shifts the curve for trithionate slightly downward but yields an error of less than +I%;alternatively greater than 10 ppm of tetrathionate lowers the
I
C
0
02
04
06
08
10
-Potential
12
V
(VI
I4
16
18
20
AgiAgCI)
Figure 3. Differential pulse polarogram: (A) thiosulfate produced M S4062through sulfitolysis of 4 X IO-' M S40e2-;(B,C) 4 X and S30e2- in CsCI-TMC-EtOH supporting electrolyte solution, respectively: drop time, 0.5 s; modulation amplitude, 50 mV; scan rate, 5 mV s-I.
curve even more, and the subsequent error in the trithionate determination increases to more than +5 % . In such cases, dilution of the sample solution will avoid this error. Presumably, this error arises from preferential adsorption of tetrathionate ions onto the mercury electrode surface, which reduces the reduction current of trithionate ions (16). Overlapping of reduction waves for both anions can be ruled out as an error source, because, if this were the case, the calibration curve would shift upward and a negative error would be induced. Pentathionate. According to Zhdanov (34),pentathionate gives three polarographic waves among which the main diffusion wave shows a pH-dependent half-wave potential and a slightly irreversible process. It also exhibits a current minimum which depends on the concentration and the type of cations present in supporting electrolytes. In CsC1-TMCethanol solution, pentathionate shows a broad wave at a half-wave potential of -1.2 V (vs. Ag/AgCl) and overlaps seriously with the tetrathionate wave. This is the reason that the procedure for polythionates was separated into two subprocedures, depending on the concentration of pentathionate. Therefore, if pentathionate is present at more than a tenth of the concentration of tetrathionate, we must select another supporting electrolyte in order to obtain good polarographic separation of pentathionate from tetrathionate. In this respect, phosphate buffer (pH 6.5) best suppressed the minimum wave from tetrathionate and gave a good base line for pentathionate (Figure 5). The difference in half-wave potential between tetrathionate and pentathionate was more than 300 mV and the pentathionate peak at -0.50 V (vs. Ag/AgCl) was
1598
.
ANALYTICAL CHEMISTRY, VOL. 56. NO. 9. AUQUST 1984
+ (x - 3)S032-= (x -
Table 111. Polarographic Determination of Tetra-, Penta-, and Hexathionates after Sulfitolysis: S,OBZ3)Szot-
+ SaOg2-
(a) Analyses of ThionatesO through Determination of Trithionate (pg)b S40;-
CS*W
5602-
S5002-
added
found
added
found
added
found
added
found
21.9 65.8 111.7
21.8 f 0.7 64.7 f 1.6 112.0 k 1.8
28.3 85.2
28.6 i 0.1 85.6 f 2.3
28.5 85.6
28.6 i 2.9 83.6 i 1.2
15.3 77.5
15.4 f 0.3 78.0 f 1.0
(b) Analysis of Thionates through Determination of Thiosulfate (pM as thiosulfate)c
S20$- + S406*- t
S002-
S50,2-
s40;-
5506'- t s606'found
added
found
added
found
added
found
added
4.00 20.0 40.0
4.10 i 0.2 19.8 f 0.2 39.0 f 0.3
39.8
39.0 i 0.1
60.0
62.5 i 0.2
30.9 80.2
31.1 i 0.5 80.6 i 0.5
Mean deviation from three replications. Microgram as trithionate. Mean deviation from three replications. 3
and Physics", 58th ed.). The pH 5.0 citrate mobile solution for HPLC and phosphate buffer at pH 6.5 were used as the supporting electrolyte in our procedure. Because hexathionate is similar in stability to pentathionate, the half-wave potential of the main reduction wave for hexathionate anion also varied from -0.3 to -0.8 V SCE with increasing pH of the solution (17) and shifted with increasing concentration of hexathionate toward more negative potential values. Analytically, the hexathionate anion exhibits a sharp primary reduction wave which is detectable at concentrations down to M. The M. calibration curve of this species is linear up to Total Thionates. The analytical scheme presented here depends upon determining the total amount of polythionates as the basis for the analysis of trithionate. Koh and Taniguchi (5)and Moses et al. (40) used the following reaction to obtain both total thionates and the average sulfur content of the thionates:
5,062-
3 25 5!J
0
7.5
10.0
Concentration
12.5
15.0
17.5
x10-5 M
Flgure 4. Polarographic calibration curves for trithionate in CsCITMC-EtOH mixture and the effect of coexisting tetrathionate on its M S40t, ;with 0-4.5 X determination: curves 1 and 2, SO (open diamond; filled circle); curve 3, SO , -; with 1.25 X IO4 M S40;(filled squares). Curves A-D show wave height of tetrathionate as a function of trlthionate concentration: (A) 2.5 X lo-' M (B) 5.0 X IO6 M S40e2-,respectively (all open M; (D) 1.25 X M; (C) 7.5 X circles).
so sharp that it was completely separable from the tetrathionate wave at the peak potential of -0.17 V. In addition, the detection limit of this anion under the studied condition was as low as lo-' M. The calibration curve for pentathionate is linear up to M. Hexathionate. Literature on the polarogram of hexathionate is rare, probably because of difficulties in preparing pure hexathionate salt. Cavallaro et al. (12) showed a polarogram of the hexathionate anion in a solution of 0.5% Rochelle salt, 0.1 N barium chloride with 0.01% gelatin at -0.66 V (vs. SCE). This supporting electrolyte, however, precipitates barium tartarate, because the solubility of this compound is about 0.04% at 20 "C ("Handbook of Chemistry
+ ( X - 3)CN- + 20H- = 504'- + 5203'- + HzO + ( X - 3)SCN- (3)
where x = 4,5 and 6. The former authors (5) determined thiosulfate colorimetrically and the latter (40), chromatographically, but neither determined trithionate, because cyanolysis of trithionate proceeds only through boiling of the reaction mixture (7) and also it is insensitive to UV radiation (41).
Thus, we have used sulfitolysis in our procedure. Sulfitolysis of polythionates produces trithionate whose molar ratio to the thionates in the original solution is 1:l (cf. eq 1). The recovered trithionate was determined polarographically together with the preexisting trithionate in the sample solution. Contrary to the procedure for thiosulfate determination, the procedures proposed for the trithionate analysis do not include masking of excess sulfite in the reaction mixture by formaldehyde, because the adduct, H2C(OH)2(S03)-,gives a polarographic wave at a half-wave potential of -1.58 V (vs. Ag/AgCl), which is close to the potential of trithionate at -1.31 V. Trithionate is recovered stoichiometricallyby polarography without adding formaldehyde, if measured within 20 min after completion of sulfitolysis (Table 111). Longer measurement times brought slightly higher values than expected, probably because of the production of trithionate from reaction between thiosulfate and unmasked sulfite according to the following equation:
+
+
Sz032- 4S032- 6H+ = 2S3062-+ 3H20
(4)
Data in Table I11 show that the recovery of trithionate from
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984
Table IV. Analysis of Mixture of Thiosulfate and Polythionates
(pg)'
s*o*-
s,o,1-
52032-
1599
s,o,2-
s5°6z
added
found
added
found
added
found
added
found
added
found
1.06 2.66
1.01 + 0.05 2.62 f 0.10
0.89 4.39
0.76 f 0.13 4.12 f 0.57
1.13 5.86
1.33 f 0.16 6.38 i 0.31
1.22 6.09
1.18 0.12 5.95 0.11
*
0.13
0.13 f 0.01 6.76 f 0.14
6.71
Average deviation from two replications. A
J-L 0
-02
-04
-06
-08
Potentia\,V (w AglAgCl)
Flgure 5. Differentlal pulse polarogram of pentathionate with tetrathionate: supporting electrolyte, 1 M K2HP04-Na2HP04(pH 6.5); drop time, 0.5 s;modulation amplitude, 50 mV; scan rate, 5 mV 8';mercuy M S4Oe2-,(B)5 X IO-' M S50e2-. drop size, medium: (A) 5 X
original thionate is excellent without using formaldehyde as a masking reagent for excess sulfite in the sample mixture if analyses are made within 20 min. Total Amounts of Thiosulfate, Tetra-, Penta-, and Hexathionates. Polarograms of thiosulfate as a sulfitolysis product gave a slightly concave calibration curve. Full deaeration of the sample mixtures was required to avoid interference by dissolved oxygen which gave a reduction wave at -0.05 V (vs. Ag/AgCl). Table I11 shows satisfactory recoveries of thiosulfate from tetra-, penta-, and hexathionates through sulfitolysis. It also shows that molarities of thiosulfate being recovered from the mixture of thiosulfate and polythionates were reasonably equivalent to those expected from eq 1. Hexathionate gave a slight excess recovery (+4.2%). This error may come from impurities such as higher polythionates in the hexathionate stock solution. The chromatogram of the hexathionate stock solution showed three small peaks after the hexathionate peak. The polarographic waves of these impurities have the same half-wave potentials as those of penta- and hexathionates, but they are likely to have higher sensitivities at the dropping-mercury electrode, because hexathionate showed a considerable decrease in the polarographic reduction current when the impurities were removed by HPLC, although they were present at less than 2.5% with hexathionate. We checked the recovery of hexathionate from the stock solution which was not purified by HPLC using a purified, dilute, standard hexathionate solution. The stock solution was much too concentrated to be purified by the commercially available analyzing column. Therefore, hexathionate is unavoidably recovered in slight excess. Recovery of Thiosulfate and Polythionates from Mixtures. Table IV shows the recoveries of each anion from the mixture of thiosulfate and tri-, tetra-, penta-, and hexathionates using our procedures. Tri- and tetrathionates, calculated by methods (vi) and (vii) in part I11 of the polarography section
of this paper, exhibit considerably larger errors due to those produced in determining other anions. If higher polythionates, such as penta- and hexathionates, are present in larger amounts than thiosulfate and/or lower polythionates, the errors for the higher polythionates will result in accumulated larger errors for the values for tri- and tetrathionates, because, as predicted from method (vi), the higher polythionate errors will be increased by the stoichiometricmutliplying factors for penta- and hexathionates. This will not be the case in most applications, however, because pentathionate, hexathionate, and higher polythionates usually exist only in small proportions relative to thiosulfate, sulfite, or tri- or tetrathionate (2, 42); therefore, errors for tri- and tetrathionates in actual sample analysis will be smaller than those in this study.
ACKNOWLEDGMENT The authors thank Sumaria I. Mohan Neil of Applied Science Co. for technical assistance in HPLC. We are grateful for comments by J. Jordan and J. Yakupkovic of the Department of Chemistry, The Pennsylvania State University, and by two anonymous reviewers. Registry No. S4062-, 15536-54-6; S502-, 15579-16-5; S6O2-, 31294-89-0; S203'-, 14383-50-7; S3O6'-, 15579-17-6.
LITERATURE CITED
(28) (29) (30) (31) (32) (33)
Schmidt, J. W.; Conn, K. "Abatement of Poiution from Mine Wastewaters"; Proceedings of the First Annual Meeting of the Canadian Mineral Processors, 1969. Goldhaber, M. 8. Am. J. Scl. 1983, 283, 193-217. Szekeres, L. Talanta 1974, 2 1 , 1-44. Koh, T.; Iwasaki, I.Bull. Chem. SOC.Jpn. 1965, 3 8 , 2135-2138. Koh, T.; Taniguchl, K. Anal. Chem. 1973, 45, 2018-2022. Koh, T.; Taniguchi, K.; Miura, Y.; Iwasakl, I . Nippon Kagaku Zasshi 1979, 348-353. Kelly, D. P.; Chambers, L. A.; Trudinger, P. A. Anal. Chem. 1969, 41, 898-901. Mizoguchi, T.; Okabe, T. Bull. Chem. SOC. Jpn. 1975, 48, 1799-1805. Nor, Y. M.; Tabatabai, M. A. SollScl. 1976, 122, 171-178. Furness, W.; Davies, W. C. Analyst (London) 1952, 77, 697-707. Murayama, S. Nippon Kagaku Zasshi 1953, 349-352. Cavallaro, L.; Bighi, C.; Pancaidi, G.; Trabanelli, 0. Ann. Chlm. (Rome) 1958, 48, 466-477. Puiidori, F.; Bighi, 0.; Borghesanl, G.; Pedriaii, R. Ann. Chlm. (Rome) 1966, 56, 1562-1581, Zezula, I.Collect. Czech. Chem. Commun. 1968, 33, 18-25. Zezula, I.Collect. Czech. Chem. Commun. 1968, 33, 2327-2332. Zezula, I.Collect. Czech. Chem. Commun. 1969, 3 4 , 355-363. Zezula, I. Collect. Czech. Chem. Commun. 1970, 3 5 , 2355-2366. Skorsepa, J. Collect. Czech. Chem. Commun. 1970, 3 5 , Zezula, I.; 1660- 1670. Tuovlnen, 0. H.; Nlckoias, D. J. D.Appl. €nvlron. Mlcrohlol. 1977, 33, 477-479. Chapman, J. N.; Beard, H. R. Anal. Chem. 1873, 45, 2268-2270. Waikoff, A. W.; Larose, R. H. Anal. Chem. 1975, 47, 1003-1008. Reeve, R. N. J. Chromatoor. 1979. 177. 393-397. Stamm, H.; Goehring, M.; Feldmann, U. 2.Anorg. AI@. Chem. 1942, 250, 226-228. Goehring, M.; Feidmann, U. 2. Anorg. Al/g. Chem. 1848, 257, 223-226 -_ . -_ -. Schmldt, M.; Sand, T. J. Inorg. Nucl. Chem. 1964, 26, 1185-1188. Naito, K.; Hayata, H.; Mochizukl, M. J. Inorg. Nucl. Chem. 1975, 37, 1453- 1457. Meites, L. "Polarographic Techniques"; 2nd ed.; Interscience: New York, 1965. Wolkoff, W.; Larose, R. H. J. Chromatogr. Scl. 1976, 14, 353-355. Lyons, D.; Nickless, 0. "Inorganic Sulfur Chemistry"; Eisevler: New York, 1968; Chapter 14. Kolthoff, I. M.; Mllier, C. S. J . Am. Chem. SOC. 1941, 63, 1405-1411. Noel, D. L. J . Tech. Assoc. Pulp Paper Ind. Korea 1978, 61, 73-76. Renard, J. J.; Kubes, 0.; Boiker, H. I. Anal. Chem. 1975, 47. 1347-1352. Tuovinen, 0. H. Talanta 1978, 2 5 , 408-409.
1600
Anal. Chern. 1884, 56, 1600-1603
(34) Zhdanov, S. I, I n “Encyclopedia of Electrochemistry of Elements”; Bard. A. J., Ed.; Plenum: New York, 1975; VoI. 4, Chapter 12. (35) Nriagu, J. 0.; Coker, R. D.: Kemp, A. L. K. Limnol. Oceanogr. 1979, 24, 383-389. (36) Makhlja, R.; Hltchen, A. Talanta 1978, 25, 70-84. (37) Zezula, I. Chem. Lis@ 1953, 47, 1303-1308. (36) Subrahmanya, R. s. Proc,-Indkn Acad. sci., Sect. A 1955, 42, 267-278.
(39) WeaSt, R. C.3 Ed. ”Handbook Of Chemistry and Physics”, 58th Ed.: CRC Press: Boca Ratan, FL, 1977; p 8-94, (40) Moses, C. 0.; Nordstrom, D. K.; MiUs, A. L., submitted to Talanta.
(41) Schmidt, M.; Sand, T. J . Inorg. Nucl. Chem. 1964,26,1173-1177. (42) Iguchi, A. Bull. Chem. SOC.Jpn. 1958, 3 1 , 597-605.
RECEIVEDfor January 30,1984. Accepted April 13,1984. This research Was supported by the U.S. Bureau of Mines under Contract No. J O 100065 to L. M. Cathles and H. L. Barnes and by the ore it^ ~~~~~~~hsection of The pennsylvania State University.
High-Performance Thin-Layer Chromatography of Gibberellins in Fermentation Broths Patricia Holt Sackett Abbott Laboratories, 14th Street and Sheridan Road, North Chicago, Illinois 60064
A method has been developed for high-performance thin-layer chromatography (HPTLC) that yields quantitative assay values for gbberellns A, and A, f A,, comblned, In fermentation broths. The llnear ranges for both measurements were from 0 to about 200 pg/mL. Preclslon was 3-4% for repilcates of a single sample, and assay values calculated by two analysts agreed wHhln 6% for A,. Recoveries were sllghtly hlgh for A,. Agreement was good when 14 fermentation broth samples were assayed by both HPTLC and HPLC methods. Resolution from potential interferences, notably glbberellenlc acid, Is documented.
Separation and quantitation of the gibberellin family of plant growth regulators have received much attention in the literature (2-4). The small differences in functional groups between the various derivatives place high demands on the specificity and resolving power of any chromatographic technique. With the advent of high-performance liquid chromatography (HPLC), interest shifted from traditional thin-layer chromatographic (TLC) methods, and focused instead on the newer technique. For complex sample matrices (fermentation broths, plant tissues), column fouling could occur, requiring extensive sample cleanup or frequent replacement of expensive columns. Sample throughout was limited by the run time of a single sample and possible column wash and reequilibration steps between samples. The recent appearance of 10-pm high-performance thinlayer chromatography (HPTLC) plates, as well as spotting and densitometer instrumentation designed with their use in mind, makes this technique a viable alternative for those without extensive experience with TLC. The increased efficiency of these plates allows separation of more complex mixtures and similar compounds in a shorter time. In this laboratory a method was needed that would allow quantitation of selected gibberellins in as many as 50-100 samples of fermentation broth per week. The following describes a system capable of separating gibberellic acid (AS, 1) from related compounds A4 (2) and A7 (3). Because quantitation of the A3 species, the predominant component in commercial formulations, was deemed of primary importance, separation of the A4 and A7 derivatives was not stressed. Mobile phases capable of separating these closely related species generally contain carcinogenic solvents such as benzene or potentially 0003-2700/84/0356-1600$01.50/0
I. -
2
3,
+
hazardous combinations such as chloroform acetone. The mobile phase adopted here does not resolve A4 and A7, and validation work proceeded based on the total combined concentration of the two.
EXPERIMENTAL SECTION Compounds, Solvents, and Reagents. Reagent grade ma-
terials were used unless otherwise specified. Standards and samples were diluted with methanol containing 0.1 g/2 L 4,5dihydroxy-3-(p-sulfophenylazo)-2,7-naphtha~enedisulfonicacid (DSNDA) (Eastman Kodak, Rochester, NY) acting as a visual locator. The mobile phase consisted of 4:5 (v/v) cyclohexane (Aldrich Chemical,Milwaukee, WI) + acetone and was stored over 3A molecule sieves (Aldrich). GibberellinsAS,A4,and A7 (Abbott Laboratories, North Chicago, IL) were at least 99% pure; stock solutions were prepared fresh weekly and kept refrigerated when 0 1984 American Chemical Society
