1484
Anal. Chem. 1992, 64, 1484-1489
Determination of Iodide by High-Performance Liquid Chromatography after Precolumn Derivatization Krishna K. Verma,' Archana Jain, and Archana Verma Department of Chemistry, Rani Durgavati University, Jabalpur 482001, M a d h y a Pradesh, India
Derivatlzation of iodlde Into 4-iodo-2,6-dimethyiphenoi and HPLC of lodophenol with UV detectlon has been found to be a suitable method for the sensitive detection of iodide. The precoiumn derivatlzation involved oxldation of Iodide with 2lodosobenzoateat pH 6.4 In the presenceof 2,6dimethyiphenoi. The reaction mixture was chromatographed on an octadecyisliane column using a mobile phase of acetonitriiewater, 60:40 (v/v), and detectlon at 220 nm. With an lnjectlon volume of 100 pL, the detection limit was 0.5 ng of Iodide. After complete reactlon of iodide at pH 6.4, the solutlon was adJustedto pH 1 to effect the derlvatlzation of bromide If it was also present with iodide. A moblie phase of acetonitrliewater, 4555 (v/v), was used for the simultaneous determlnatlon of Iodide and bromide. The detectlon limit for bromide was 0.2 ng. Methods have also been evolved for Iodine (precolumn reaction without using 2-iodosobenzoate) and Iodate (reduction with ascorblc acid to iodide and Its derivatlzation) and for the analysts of lodlde, Iodine, and iodate In the presence of each other. Application of the method has been made to the determlnatlonof lodlde in naturalwater, sea water, Iodized sait, milk, and pharmaceuticals. The RSD was in the range 0.4-2.9 %.
INTRODUCTION Iodide is known to be an essential micronutrient. The use of iodinated salt in the diet is the most frequent way of obtaining an additional supply of iodide. Excessive iodine intake can contribute to certain thyroid disorders in susceptible individuals whereas a deficiency of iodide in the diet produces several diseases with known symptoms.' Studies on the biosynthesis of thyroid hormones have led to the postulation of an eventual substitution of iodide by bromide, which would result in the formation of brominated or mixed brominated/iodinated thyronines with similar hormone activity. Attention has also been drawn to the role of iodide and bromide in the formation of trihalomethanes, which are cancer suspect agents, during the oxidative treatment of drinking water. Therefore, sensitive and accurate determination of iodide in biological fluids, food materials such as milk products and iodized salt, drugs, and water is of much importance in medical, nutritional and epidemiologicresearch. It would also be interesting to analyze mixtures of iodide with bromide, iodine, and iodate. Many methods based on different principles have been proposed for the determination of iodide. Trace levels of iodide have commonly been determined by their catalytic effect usually on the cerium(1V)-arsenic(II1) reaction.2~3 Other reactions catalyzed or inhibited by iodide have also been
described.435 For the analysis of iodized salt, construction of a calibration graph in the presence of the same amount of chloride as in the sample is necessary, and samples such as milk and serum, which have complex matrices, require ashing prior to the fiial determination. Microwave acid digestion of the sample, preconcentration of iodide by coprecipitation with bismuth(II1) sulfide, and neutron activation analysis have been applied to biological materials and food.6 Precipitation as silver iodide, dissolution in potassium cyanide, and determination of silver in the complex by atomic absorption spectrometry have been used for the indirect determination of iodide in a flow system.' The interference of chloride was avoided by washing the precipitate with ammonia before reaction with cyanide. However, the bromide interference was severe. Indirect reactions have also been proposed for chloride and bromide by their exchange for the anion of a sparingly soluble metal salt.s10 Halides and a number of anions react in the same manner, and the methods lack selectivity. Methods based on oxidation to iodate and spectrophotometry11J2 or formation and measurement of triiodide13 in a flow system are not sensitive; preconcentration on an ionexchanger may be a suitable choice.14 A sensitive method is ion-pair formation with methylene blue and extraction spectrophotometry;l5 brilliant green has also been used in the same way.16 Anions which also form ion pairs are several and must be removed before iodide reaction. Preconcentration was combined with oxidation of iodide to improve the atomic emission limit of detection for iodine by inductively coupled plasma;" 0.75 and 31 ng/mL for iodide and iodate, respectively. Precolumn derivatization of halides to organo-halogen compounds allows their improved separation and detection utilizing conventional HPLC equipment.lsJg The arylmercury(I1) halide method18 may not be suitably disposed to analyze samples, e.g., of sea water or iodized salt, which have (4) Garcia, M. S.; Sanchez-Pedreno, C.; Albero, M. I.; Sanchez, C. Analyst (London) 1991, 116, 653456. (5) Yonehava, N.; Yamane, T.; Tomiyasu, T.;Sakamoto, H. Anal. Sci. 1989, 5, 175. (6)Rao, R. R.; Chatt, A. Anal. Chem. 1991,63, 1298-1303. (7) Esmadi, F. T.; Kharoaf, M. A.; Attiyat, M. S. Analyst (London) 1991,116, 353-356. (8) Al-Abachi, M. Q.;Salih, E. S. Mikrochim. Acta 1987,2, 203-207. (9) Almuaibed, A. M.; Townshend, A. Anal. Chim.Acta 1991, 245, 115-119. (10) Verma, K. K.; Tyagi, P.; Ekka, M. G. P. Talanta 1986,33,10091013. (11) Al-Wehaid, A.; Townshend, A. Anal. Chim.Acta 1987,198, 4551. (12)Yaqoob, M.; Masoom, M.; T0wnshend.A. Anal. Chim. Acta 1991, 248, 219-224. (13) Kamson, 0. F. Anal. Chim.Acta 1986, 179, 475-479. (14) Carlsson, A.; Lundstrom, U.; Olin, A. Talanta 1987,34,615-618. (15) Koh, T.; Ono, M.; Makino, I. Analyst (London) 1988, 113, 945OAQ
(1) Scriba, P. C. In Thyroid Disorders Associated with Iodine Deficiency and Excess; Hall, R.; Kobberling, J., Eds.; Raven Press: New
York, 1985; p 7. (2) Rubio, S.; Perez-Bendito, D. Anal. Chim.Acta 1989, 224, 185. (3) O'Kennedy, R.; Bator, J; Reading, C. Anal. Biochem. 1989, 179, 139. 0003-2700/92/0364-1484$03.00/0
(16) Niazi, S. B.; Mozammil, M. Anal. Chim. Acta 1991,252,115-119. (17) Dolan, S.P.; Sinex, S. A.; Capar, S. G.; Montaser, A.; Clifford, R. H. Anal. Chem. 1991, 63, 2539-2542. (18) Moss, P. E.; Stephen, W. I. Anal. Proc. (London), 1985,22, 5. (19) Verma, K. K.; Sanghi, S. K.; Jain, A.; Gupta, D. J. Chromatogr. 1988,457, 345-353.
0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64,
one halide in very large excess over others. Our earlier methodlg was based on the oxidation of halide and the substitution reaction of halogen with acetanilide and could only be applied to bromide because of two difficulties: (i) the reaction of iodine and acetanilide was too slow to have any analytical value, and (ii) any iodide present was oxidized to iodate by the liberated bromine. Both of these difficulties are avoided in the following method by utilizing a suitable derivatizing reagent and proper reaction conditions, and this has enabled the development of a method for the simultaneous determination of iodide and bromide a t trace levels. EXPERIMENTAL SECTION Apparatus. Liquid chromatography was carried out with a Shimadzu (Tokyo, Japan) system, consisting of an LC-5A pump, and SIL-1A manual loop injector, a Shim-pack ODS column (particle size 5 pm; 150- X 4.6-mm LdJ, an SPD-PA variable wavelength UV detector (8-pL flow-through cell), and a C-R2AX integrator fitted with a printer-plotter. Peak areas were used for quantification. Materials. 2-Iodosobenzoicacid was synthesized by a modification of the method of Chinard and Hellerman,zOasdescribed earlier.Ig The mobile phase was prepared by mixing acetonitrile and water, 60:40 (v/v) and 4555 (v/v). Sodium 2-iodosobenzoate reagent was made by stirring 400 mg of free acid with a slight molar excess of sodium hydroxide (7.6 mL of 0.2 M sodium hyroxide) and diluting to 100 mL with water in a standard flask. It was filtered through a 0.45-pm membrane filter. A solution of 100 mg of 2,6-dimethylphenol (Aldrich) in 100 mL of mobile phase was used as halogen scavenger. The phsophate buffer contained 10 g each of KH2P04 and K2HP04in 250 mL of water and was adjusted to pH 6.4. The ascorbic acid solution was made by dissolving 100 mg of reagent in 100 mL of water. A sulfuric acid solution was prepared by diluting 2 mL of analytical reagent grade concentrated sulfuric acid to 100 mL with the mobile phase. Standards. Potassium salts of iodide, iodate, and bromide and iodine were AnalaR grade chemicals from BDH and were oven-dried before use. Diphenyl (biphenyl) as an internal standard contained 25 mg of high-purity substance (Aldrich) in 250 mL of mobile phase in a standard flask. Potassium iodide solution was made by dissolving 261.4 mg of potassium iodide in 250 mL of deionized water in a standard flask. This solution contained 800 pg/mL iodide. Potassium bromide solution contained 161.2 mg of potassium bromide in 250 mL of deionized water dissolved in a standard flask to give 400 pgimL bromide. For a solution of 350 pg/mL iodate, 107.0 mg of potassium iodate was dissolved in 250 mL of deionized water in a standard flask. To make less concentrated standards, 25-mL portions of each of above solutions were separately diluted to 250 mL in standard flasks with deionized water. An 800 pg/mL solution of iodine was made by diluting an appropriate volume of a saturated aqueous solution of iodine in deionized water and standardized by reduction, ion-pair formation with methylene blue and extraction spectr~photometry.'~ Less concentrated solutions were made by sequential dilution and stored in a cool place. Procedures. Determinution of Iodide. A 100-1000-pLaliquot of a standard solution containing 8-600 pg of iodide was mixed with 1 mL of phosphate buffer, 500 pL of internal standard, 1 mL of 2,6-dimethylphenol, and 1 mL of 2-iodosobenzoate in a 10-mL standard flask, and the solution was swirled and kept for 20 min at 21 "C. The solution was diluted to 10 mL with mobile phase (acetonitrile-water, 6040, v/v), and a 10-pL portion was injected on the HPLC column. The solvent flow rate was 1mL/ min and the column back-pressure approximated 50 kg/cm2.The 9.
(20) Chinard, F. P.; Hellerman, L. Methods Biochem. Anal. 1961, I ,
NO. 13, JULY 1, 1992
1485
column eluate was monitored by an UV detector set at 220 nm with a sensitivity of 0.04 or 0.16 AUFS. Determination of Iodine. A 100-1000-pL volume of a standard solution containing 20-800 pg of iodine was treated with 1 mL each of phosphate buffer and 2,6-dimethylphenol and 500 pL of internal standard. The solution was kept for 20 min and then diluted to 10 mL with mobile phase in a standard flask. A 10-pL portion was chromatographed as described above. Determination of Iodate. To a 100-1000-pL aliquot of standard solution containing 10-350 pg of iodate was added 1 mL of ascorbic acid, and the solution was swirled for 1-2 min. Then, 1mL each of phosphate buffer, 2,6-dimethylphenol, and 2-iodosobenzoate and 500 pL of internal standard were added. The solution was kept for 20 min and diluted to 10 mL with mobile phase in a standard flask. A 10-pL portion was injected onto the column using the chromatographic conditions as described before. Determination of Iodide and Bromide. In a 10-mL standard flask, a 100-1000-pL portion of the test solution containing not more than 5 mmol of total iodide and bromide was mixed with 1 mL each of phosphate buffer, 2,6-dimethylphenol, and 2iodosobenzoate and 500 pL of internal standard. It was shaken well and kept for 20 min. Then, 1mL of sulfuric acid was added, and the solution was shaken for 1 min and diluted to the mark with mobile phase (acetonitrile-water, 4555, v/v). A 10-pL aliquot was chromatographed using the conditions as before. Determination of Iodide i n Natural Water. A 5-mL portion of filtered (0.45-pm membrane filter) natural water, containing 12-100 pg/L iodide, was mixed in a 10-mL standard flask with 1 mL of phosphate buffer, 200 pL each of 2,6-dimethylphenol and 2-iodosobenzoate, and 100 pL of internal standard. The contents were shaken well, kept for 20 min, and diluted to the mark with mobile phase, and a 100-pLaliquot was injected onto the column. The detector sensitivity was 0.005 AUFS, and other chromatographic conditions were the same as before. Determination of Iodide i n Pharmaceuticals. The contents of the liquid sample were shaken well and their appropriate aliquot diluted to a known volume withwater. A suitable portion was subjected to precolumn derivatization reaction and HPLC analysis as described for standard iodide solutions. Determination of Iodide in Iodized Salt. A 1-mL aliquot of 10% salt solution in water was mixed with 1 mL of phosphate buffer, 500 pL each of 2,6-dimethylphenol and 24odosobenzoate, and 100 pL of internal standard. The solution was shaken and kept for 20 min, diluted to 10 mL with mobile phase, and filtered through a 0.45-pm membrane filter, and a 100-pL aliquot was chromatographed as before. Determination of Iodide in Serum. A serum sample (2 mL) was centrifuged (ca. 5000 g), and the clear supernatant was evaporated to dryness at 50 "C under a stream of nitrogen. The residue was reconstituted in a suitable volume of mobile phase. A known aliquot was subjected to precolumn derivatization and HPLC analysis as described for standard iodide solution. Determination of Iodide in Milk and Milk Powder. A 2-mL volume of milk (milk powder was reconstituted with an appropriate amount of water) was mixed with 4 mL of acetonitrile and centrifuged (ca. 5000 g),and the clear supernatantwas evaporated to dryness at 50 "C under a stream of nitrogen. The residue was reconstituted in an appropriate volume of mobile phase and a suitable portion was subjected a precolumn derivatization and chromatographic analysis was described for standard iodide solutions.
RESULTS AND DISCUSSION The present methods for the determination of iodide and bromide are based on their precolumn oxidation to the corresponding halogen and its reaction with an aromatic compound to give a halogenated derivative which is determined by HPLC. Selection of the Oxidizing Agent. Many oxidizing agents such as permanganate?l peroxymonosulfate,22 and chloram(21) Utsumi, S.; Kotaka,M.; Isozaki, A. Bumeki Kagaku 1986,34,81. (22) Dobolyi, H. F. Anal. Chem. 1984,56, 2961-2963.
1486
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992
Scheme I
solutions which contain both iodide and bromide, iodide is first derivatized by reaction at pH 6.4, and then the pH was adjusted to about 1 to allow bromide derivatization. The derivatives are stable for several days. As expected, iodate did not form any iodophenol under the proposed derivatization condition for iodide. For its reaction (eq iii), iodate was reduced to iodide with ascorbic acid before subjecting it to precolumn reaction. Since the reduction of
IO;
x
=
I or B,
ine-TZ3~24have been tried for single halides. However, these cannot be used in the present precolumn reaction since any iodide present is converted to iodate either by the oxidant or by the in situ produced bromine. Iodate has been found not to undergo subsequent aromatic substitution and thus escapes determination. There was, therefore, the need of an oxidizing agent which could sequentially produce halogens under properly controlled reaction conditions, the formation of iodine occurring first to avoid iodate formation. The selectivity of 2-iodosobenzoicacid as an oxidizing agent has already been demonstrated.2529 Its redox potential at 25 "C was found to be 1.21 V a t pH 1,1.08 V at pH 2,0.53 V at pH 4, and 0.48 V at pH 7.2s Therefore, only iodine is produced in neutral or feebly acidic solutions while bromine is formed in moderately acidic medium. Selection of the Halogen Scavenger. The purpose of these reagents is to utilize halogens produced in an oxidation step to give halo derivatives which are easily separable and sensitively detectable. Attention was paid to the formation of only one isomer of a derivative in order to avoid more than one chromatographic peak for each analyte anion. This could be achieved by using a properly substituted aromatic compound which has only one available active position in the nucleus for electrophilichalogenation. Unfortunately, organic substrates which can readily undergo both iodination and bromination are not many; usually the former reaction is too slow to have any analytical use. Among the various classes of compounds tried, only aromatic amines and phenols responded for quick halogenation. However, amines tend to form oxidation products, either with halogen or the oxidant used for halide oxidation and were not used. 2,6-Dimethylphenol has been found to be a compound of choice. Its iodination was negligible in acidic medium but fairly rapid in neutral solutions, and bromination was almost instantaneous in both media. 2,6-Dimethylphenol did not show any reaction with 2-iodosobenzoic acid in either media. Chemistry of derivatization. The reactions given in Scheme I occur during the precolumn derivatization of iodide and bromide. The halide liberated in reaction ii is again oxidized by 2-iodosobenzoate and this sequence of oxidation and halogenation reactions continues until all halide has been converted into 4-halo-2,6-dimethylphenol.Over a pH range of 5-7, when there is no oxidation of bromide, both iodide oxidation and iodination of 2,6-dimethylphenol occur rapidly; a pH of 6.4 was selected as optimum. The bromide was derivatized over a pH range 0-3; pH l was selected. In test ~~
~
(23) Basel, C. L.;Defreese, J. D.; Whittemore, D. 0.Anal. Chem. 1982, 54, 2090. (24) Anagnostopoulou, P. I.; Koupparis, M. A. Anal. Chem. 1986,58, 322. (25) Verma, K. K.; Gulati, A. K. Tatanta 1983, 30, 279-281. (26) Verma, K. K.; Bose, S. Anal. Chim. Acta 1974, 70, 227-228. (27) Verma, K. K.; Bose, S. Analyst (London) 1975, 100, 366-367. (28) Verma, K. K. Talanta 1982,29, 41-45. (29) Verma, K. K.; Gupta, A. K. Talanta 1982, 29, 779-784.
+ 3C6H,06
-
I- + 3C,H,06
+ 3H,O
(iii)
iodate occurs only in acidic medium, ascorbic acid served as a reagent of choice for reduction and maintaining feebly acidic condition. Any excess of ascorbic acid from iodate reaction is subsequently oxidized by 2-iodosobenzoateto avoid halogen reduction by ascorbic acid. The derivatization of iodine had been accomplished in two ways. In the absence of 2-iodosobenzoate, only 1 mol of 4iodo-2,6-dimethylphenol was formed for every mole of iodine by reaction ii. However, in the presence of iodosobenzoate, the iodide liberated in reaction ii was also oxidized to iodine by reaction i, and thus 2 mol of iodophenol derivative were formed for each mole of iodine. Any iodide already present with iodine was also derivatized in the presence of iodosobenzoate. This principle has been used for the analysis of mixtures of iodide and iodine by derivatization of two separate but equal portions of the test solution and then HPLC. Mixtures of iodide, iodine, and iodate were analyzed for their iodideliodine content as before, when iodate did not interfere, and then iodate was determined on the third aliquot of sample after its reduction to iodide with ascorbic acid. The halogenated phenols are toxic, and their solutions should be cautiously disposed of. Validation of Derivatization. The chromatographic peaks for derivatized iodide and bromide were identified by comparison of their retention time with those of the authentic substances under the same chromatographic conditions. These compounds of corresponding retention time had identical UV and IR spectra. The quantitative nature of derivatization reaction was confirmed by HPLC and titrimetric methods. In the former methods, equal molar masses of authentic 4-halo-2,6-dimethylphenol and of halides (after derivatization) were chromatographed when their peak areas were within 1-2% for bromide and 2-3% for iodide. The titrimetric method was based on the reaction of a known quantity of iodide or bromide with a measured excess of 2iodosobenzoate at the corresponding pH of 6.4 and 1for iodide and bromide, respectively, and in the presence of 2,6-dimethylphenol. Halogen produced in the oxidation step was scavenged by phenol, reactions i and ii, and thus there was a net consumption of the oxidant. The remaining 2iodosobenzoate was evaluated iodometrically using standard sodium t h i o ~ u l f a t e .A~consumption ~ of 1mol of 2-iodosobenzoate was found for every mole of iodide or bromide, and it agreed with the value obtained on combining reactions i and 11.
ChromatographicConditions. Preliminary HPLC studies were conducted on a CIS reversed-phase column with a mobile phase of methanol and water in various proportions. With a 60:40 (v/v) methanol-water composition, the elution of 2-iodo- and 2-iodosobenzoic acid was fast and occurred first but halophenol derivatives were eluted as unseparated broad peaks and there was a positive baseline drift in the chromatogram. In subsequent studies, acetonitrile was used in place of methanol. Sharp peaks were obtained with an acetonitrile-water (6040, v/v) mixture having a flow rate of 1 mlimin. However, the halophenol derivatives were not resolved, k' = 1.3 and 1.5for derivatized bromide and iodide, respectively (Figure 1A). A lower solvent flow rate did not
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 1. 2
Table I. Determination of Iodide, Bromide, Iodate, and Iodine in Their Standard Solutions analvte concn taken concn found % RSD
I 2 3 I
iodide 1.2
I
bromide
-
0
2
4
6
0
iodate
0
2
4
6
1487
I
l
,
,
8
0
5
10
>
l
15
20
T I M E . MIN
Flgun, 1. Chromatogramsof precolumn-derivatized iodide and bromide. Condltlons: moblle phase A and B, acetonltrile-water, 60:40 (v/v); mobile phase (C) acetonltrile-water, 4555 (v/v); flow rate, 1 mL/min; column, ODs (5-pm particle size, 150- X 4.6-mm Ld); detectionat 220 nm. Peaks: (1) and (2) 2-iodo- and 2-lodosobenzoate; (3) 2,6dimethylphenol; (4) 4-bromo-2,6dlmethylphenol; (5) 4-iodo-2,gdimethylphenol; (6) diphenyl (internal standard).
improve the separation. Since the analysis was complete in about 6.5 min, this solvent composition was used when only iodide or bromide was being determined (Figure 1B). For the simultaneous determination of both halides, the acetonitrile-water (45:55, v/v) mixture was used and it resulted in complete resolution of halophenol peaks, k’ = 3.7 and 4.6 (Figure 1C). Attempts were also made to study the influence of incorporating tetrahydrofuran (THF) in the mobile phase. Acetonitrile-THF-water (3510:55, v/v), with a flow rate of 1mL/min, produced a baseline separation of halophenols, k’ = 3.3 and 4.2. However, solutions of halophenols in THFcontaining solvents soon developed a yellow color in diffused light possibly due to some photoreaction. The use of THFcontaining solvents is not recommended for the liquid chromatography of halophenols. Optimum absorption for halophenols was observed at 220 nm. Calibration Graph and Detection Limits. The limit of detection (signal-to-noiseratio 3:l) of the method using iodide and bromide standards was 0.5 ng of iodide and 0.2 ng of bromide in a 100-pL injection volume. A valid calibration was obtained between the amount of analyte and the peak area of its precolumn derivative from the detection limit up to the following amounts injected (correlation coefficient, r; n = 10): iodide, 800 ng (0.9992); bromide, 400 ng (0.9999). After relative weight correction, the calibration graph for iodide can be used for iodine and iodate determination since equal mole concentrations of iodide, iodine (in the absence of 2-iodosobenzoate), and iodate produce the same peak area on HPLC; the calibration graph was linear up to 700 ng (0.9989) and 400 ng (0.9996) of iodine and iodate, respectively. Results are given in Tables I and I1 for the determination of iodide, bromide, iodate, and iodine either present separately or present in their various combinations. In precision studies, five or six aliquots of the same sample were separately derivatized and one injection each was made. Applications. The present method has been applied to determine iodide in a variety of matrices. Results are given
iodine
8.21 22.32 38.54 52.16 125.16 378.1 529.0 654.2 790.7 4.15 18.66 37.18 55.72 162.3 249.9 338.4 390.2 9.75 20.36 33.81 46.50 120.6 213.2 260.9 301.4 346.2 19.62 42.79 84.50 168.3 250.5 336.7 412.5 549.2 682.1
8.15 22.40 38.65 51.82 124.0 380.6 523.8 646.2 784.5 4.18 18.71 37.44 56.31 160.9 251.8 337.4 386.7 9.62 20.45 33.74 46.63 119.5 214.9 262.1 298.6 343.8 19.54 43.52 85.71 167.8 253.1 332.9 418.7 543.6 677.4
1.5 1.2 1.1 1.1 1.2 1.5 1.6 1.6 1.8 0.8 0.8 0.4 0.5 0.6 0.6 0.9 1.0 1.4 1.2 1.2 1.0 1.0 1.2 1.5 1.5 1.6 1.5 1.4 1.0 1.0 1.2 1.5 1.6 1.8 1.8
a The results, given as pg of analyte/lO mL,are the average of six determinations.
in Table I11 for the analysis of laboratory made or commercially available pharmaceuticals. Synthetic fresh water samples of the relative ionic composition as used previously30 and containing as low as 13 pg/L iodide were analyzed (Table IV). Due to low levels of iodide content, a larger volume of natural water sample was taken for precolumn derivatization and completed with acetonitrile to match the solvent composition in the injection solution near to that of mobile phase. In Table V, results are given for the analysis of serum samples spiked with known quantities of iodide. Food samples of iodized salt and milk were analyzed for their iodide content, and the results are summarized in Tables VI and VII, respectively. In all cases, the results were within permissible errors and had close agreement with those obtained by previously evaluated methods.15*31%2The present method is thus selective and widely applicable to determine iodide in different natural samples. Luckas33 proposed a scheme for differentiating between iodide, iodine, and iodate in salts using HPLC and with UV detection. Three analyses were necessary, first to determine free iodide (without any sample pretreatment), the second to evaluate the sum of iodide and iodine (after reduction of the sample with hydrazinium sulfate), and the third to obtain total iodine species (after reduction of the sample with stannous chloride). Nevertheless, this scheme is liable to produce erroneous results because on-line reduction of iodine has been (30) Lundstrom, U.; Olin, A.; Nydahl, F. Talanta 1984, 31, 45-48. (31) Funazo, K.; Tanaka, M.; Shono, T. Anal. Sci. 1987,3,41-44. (32) Lambert, J. L.; Hatch, G.I,.; Mosier, B. Anal. Chem. 1976,47,
915-916. (33) Luckas, B. Dtsch. Lebemm-Rundsch, 1986,82,357.
1488
9
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992
Table 11. Determination of Synthetic Mixtures of Iodide, Bromide. Iodine. and Iodate concn found concn taken I I1 I11 I I1 I11 IBr11.25 29.36 40.28 63.71 89.06
61.33 45.12 29.34 15.80 6.77
I-
12
10.55 19.76 28.22 36.70 49.51
80.21 64.32 52.46 36.29 25.41
11.32 (1.3) 29.71 40.80 (1.1) 63.18 89.65 (1.0)
61.52 (0.8) 44.63 29.78 (0.6) 15.65 6.70 (0.7)
10.36 (1.3) 20.1 (1.1) 28.50 36.48 49.81 (1.2)
80.56 (1.1) 64.08 (1.2) 51.76 36.50 25.26 (1.3)
12.34 22.91 30.72 43.26 55.61
46.89 39.74 28.56 15.70 9.22
I-
I2
9.21 25.60 32.29 15.22 21.06
15.73 10.64 25.71 36.15 19.81
12.49 (1.0) 23.20 30.42 (1.2) 43.71 55.26 (1.0)
46.70 (1.3) 39.52 28.38 (1.0) 15.62 9.19 (1.3)
8.96 (1.3) 25.84 31.74 (1.2) 15.43 21.25 (1.2)
15.87 (1.0) 10.58 25.99 (1.3) 36.52 19.70 (1.3)
10.00
10.94
1.2
4f
13.00
12.85
1.1
58
15.00
15.19
1.3
22.69 (1.8) 30.90 15.22 (1.6) 13.81 39.75 (1.6)
10.85 (MB) 10.08 (HPLC) 10.51 (LCV) 12.91 (MB) 13.07 (HPLC) 13.22 (LCV) 15.17 (MB) 15.21 (HPLC) 14.88 (LCV)
The results, given in pg of iodide/mL, are the average of five determinations. The abbreviations used for comparison methods are MB = methylene blue-iodide ion-pair formation and spectrophotometry,15 HPLC = derivatization to 4-nitrobenzyl iodide and HPLC,3I LCV = leuco-crystal violet oxidation and spectrophotometry.32 c Also contains theophylline (5.33 mg). Also contains aminophylline (1mg) and sodium saccharin (0.3 mg). e Also contains theophylline (6 mg). f Also contains aminophylline (2 mg). 8 Also contains sodium saccharin (2 mg). 0
reported to occur under HPLC ~onditions.3~The present method of precolumn derivatization to 4-iodo-2,6-dimeth(34) Buchberger, W. J. Chromatogr. 1988, 439, 129-135.
2.8 2.2 1.8 1.7 1.7 1.8
% RSD
8.31 16.42 29.55 38.29 46.30
2.0 1.8 1.5 1.4 1.5
8.50 16.35 29.36 38.42 46.87
The results, given as pg of iodide/mL of serum, are the average of five determinations.
Table 111. Determination of Iodide in Pharmaceutical Preparations concn concn determined determined nominal by present by comparison formulation concn method % RSD methodb Commercial 1C 8.66 8.49 1.0 8.52 (MB) 8.54 (HPLC) 8.60 (LCV) 2d 10.00 10.80 1.1 10.64 (MB) 10.38 (HPLC) 10.97 (LCV) 3e
12.71 25.80 38.67 46.25 59.62 70.36
Table V. Determination of Iodide in Serum sample no. concn added concn found 1 2 3 4 5
a The results are given in pg of analytei10 mL. Values followed by % RSD in parentheses are the average of five determinations and the rest are the average of three determinations.
Laboratory Made
12.82 25.34 38.15 46.92 59.09 71.12
3.715.30
10322.88 30.52 15.40 13.69 40.12
1 2 3 4 5 6
L2 The results, given as pg of iodide/lOOOmL,are the averageof five determinations. The "standard composition, 0.005 N"contains ions (mequiv/L): calcium, 3.175; magnesium, 0.870; sodium, 0.785; potassium, 0.170; chloride, 0.5; sulfate, 0.785; hydrogen carbonate,
10~-
I-
Table IV. Determination of Iodide in Natural Water solution no. concn added concn found % RSD
Table VI. Determination of Iodide in Iodized Salt concn concn determined determined by present by comparison samules method % RSD methodb Laboratory Made 1 C 41.1 1.0 40.7 (MB) 2d 61.8 0.8 62.1 (HPLC) 3e 84.5 1.0 84.8 (LCV) Commercial 4 5 6
53.8 73.0 89.6
1.1 1.0 0.8
53.2 (MB) 72.1 (HPLC) 89.2 (LCV)
The results, given as pg of iodide/g of iodized salt, are the average of five determinations. The abbreviations used for comparison methods are MB = methylene blue-iodide ion-pair formation and spe~trophotometry,~~ HPLC = derivatization to 4-nitrobenzyl iodide and HPLC,3l LCV = leuco-crystal violet oxidation and spectrophot ~ m e t r y .Also ~ ~ contains potassium bromide (lOOmg/g)and sodium sulfate (50 mg/g). Also contains potassium iodate (20 mg/g). e Also contains sodium hydrogen carbonate (25 mg/g)and magnesium sulfate (15 w i g ) .
ylphenol and ita HPLC is free from these shortcomings, since iodine itself is never injected, and has been successfully used to analyze such mixtures. Interferences. The interference of a number of different ions has been studied by spiking 40 pg/mL of iodide with known quantities of foreign materials and analyzing it by the present method. Any organic matter that also undergoes iodination, such as aniline and phenol, interfered severely and should be removed, e.g. by passing the test solution through a column packed with nonpolar porous polystyrene-divinylbenzene resin (Amberlite XAD-2) before precolumn derivatization.12 Reducing agents could react either with the liberated iodine or 2-iodosobenzoic acid leading to an incomplete derivatization reaction. This could be avoided by increasing the amount of 2-iodosobenzoic acid. Under the present conditions of determination, up to a &fold (m/m) excess of iron(II), sulfide, thiosulfate, sulfite, thiocyanate, nitrite, and manganese(I1) produced no greater than -3% error. Chloride, bromide, nitrate, phosphate, perchlorate, sulfate, hydrogen carbonate, calcium, magnesium, zinc, cadmium, and cobalt could be avoided when present up to
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992
Table VII. Determination of Iodide in Milk and Powered Milk concn concn determined determined by bypresent % comparison concn % ’ sample method RSD methodb added recovered cow’s milk 1.25 1.8 1.38 (MB) 22.71 98.7 49.84 99.1 cow’s milk 1.62 2.0 1.57 (HPLC) 31.62 97.6 46.29 102.3 milk powder 5.40 1.6 5.26 (LCV) 32.60 102.0 53.31 99.1 milk powder 6.02 1.8 5.86 (MB) 20.61 99.6 42.38 102.4 a The results, given as pg of iodide/g of milk sample, are the average of five determinations. b The abbreviations used for comparison methods are MB = methylene blue-iodide ion-pair formation and epe~trophotometry,~~ HPLC = derivatization to 4-nitrobenzyl iodide and HPLC,3l LCV = leuco-crystal violet oxidation and
spectroph~tometry.~~
1000-fold (m/m) excess over iodide, the error being not more than 2 7%. In case of any turbidity, the precolumn derivatized solution was filtered through a 0.45-pm membrane filter before injection. Iron(III), copper(II1, thallium(I), lead, and mercury(I1) were masked with ethylenediaminetetraacetic acid (disodium salt, EDTA) and tolerated up to 50-foldexcess, the maximum error being 3 % . Up to 100-fold excess of ammonium ion did not interfere.
1489
conditions. With a combination of the suggested schemes of derivatization with or without the presence of 2-iodosobenzoic acid and after reduction with ascorbic acid, it is possible to analyze iodide in the presence of iodine and iodate, and also in the presence of bromide. The obvious drawback is the necessity to perform three independent determinations. The rival procedures of ion chromatography could analyze simultaneously both iodide and iodate among other ions. For samples with a diversity of anionic constituents, the almost universal response of conductometric and refractive index detectors can be a disadvantage if chromatographic resolution is inadequate. Electrochemical detectors are mostly used for determining halides, cyanide, and sulfide. Compared to conductometric detection, the analysis of halides by electrochemical detectors affords greater selectivity. The advantage of the present method lies in ita improved chromatographic separation and sensitive UV detection. There is a potential for the prospective use of the present procedure as a sample cleanup method in coupled HPLC-GC technique.
ACKNOWLEDGMENT Sincere thanks are due to the Council of Scientific and Industrial Research (New Delhi) for the award of a research associateship to A.J. and a senior research fellowship to A.V. Paper presented in the 5th Symposium on Handling of Environmental and Biological Samples in Chromatography at Baden-Baden, Germany, September 26-27, 1991.
CONCLUSIONS The precolumn derivatization is an attractive alternative to avoid the on-column reduction of iodine under HPLC
RECEIVED for review December 10, 1991. Accepted March 16, 1992.
