Anal. Chem. 1982, 54, 793-796
correct for the interference by simultaneously determining the amount of sulfur and phosphorus (assuming these are present predominantly as SO4 and PO4) present from the X-ray spectra of each sample.
LITERATURE CITED (1) Rolnel, N. J . Mlcrosc. 1975, 22, 261-268. (2) Lechene, C. “Microprobe Analysis as Applied to Cells and Tlssues”; Hall, T., Echlin, P.. Kaufman, R., Eds.; Academlc Press: New York, 1973; pp 351-367. (3) Rick, R.; Horster, M.; Elauer, R.; Dorge, A.; Thurau, K. ffluegers Arch. 1977. 369. 95-98. (4) Qulnton, P.’M. Micron 1978, 9 , 57-89. (5) Caflish, C. R.; Carter, N. W. Anal. Blochem. 1974, 60, 252-257. (6) Kaiser,. D.:. Sonoo-Willlams,. R.:. Drack. E. fflueuers Arch. 1974., 349.. ” 63-72. I
793
(7) Karlmark, B.; Sohtell, M. Anal. Biochem. 1974, 5 3 , 1-11. (8) Hevert, F. Pfluegers Arch. 1973, 344, 271-274. (9) Vurek, G. G.; Warnock, D. G.; Corsey, R. Anal. Chem. 1975, 47, 765-767. (10) Williams, D. D.;Mlller, R. R. Ind. Eng. Chem. Fundam. 1970, 9 , 454. (11) Maffly, R. H. Anal. Chem. 1969, 47, 273. (12) Quinton, P. M.; Earlbaum, A. Nephron 1981, 28, 58. (13) Schamber. F. “X-Ray Fluorescence Analyses of Envlronrnental Samples”; Dzubay. T. G., Ed.; Ann Arbor/Sclence: Ann Arbor, MI, 1977; pp 241-257.
RECEIVED for review August 10.1981. AcceDted December 31,1981. This work was supported by Grantsfrom the Getty cO., the cO*, and the Institutes Of (Grant No. AM00708 and AM26547).
Spectrophotometric and Titrimetric Determinations of Ascorbic Acid Naresh Kumar Pandey‘ Department of Postgraduate Studies & Research in Chemistry, University of Jabalpur, Jabalpur ---482 00 1, India
Three slmple, rapid, and accurate methods of determlnlng ascorbic acid in samples of the mlcrograrn level have been described. The ascorblc acid was determlned spectrophotometrlcaliy at 336 nm, via a decrease In absorbance In 7 X l o 4 M tetrachlorobenzoqulnone(chloranll) In 80 % acetonewater (v/v) at room temperature. Visual and potentlometrlc methods were developebd In the presence of EDTA and found to be accurate to rt0.18 to f0.45%, with a standard deviation of 0.042-0.083. Tlhe proposed methods were successfully applied to pharmacieutlcai preparations. Strong reducing agents lncludlng most 01 the thlols and serlne, glyclne, alanine, cltric, oxailc, tartaric aclds, glucose, sucrose, and maltose do not Interfere, even when present upto a 10-15 molar excess of vltamln C. Hence resoiutlon of mixtures of vltamln C and thiols Is posslble, ellmlriatlng the use of a masking agent for thiols In other methods.
Ascorbic acid (vitamin C) contains an ene-diol functional group. The many methods available for its determination have been reviewed in detail (1-15). A recent oxidimetric method (16) involves the use of chloramine-T for ascorbic acid mixtures with thiols. This method requires masking of the thiol by cyanoethylating it with acrylonitrile. The authors claim that the cyanoethylated products of thiols do not hamper the reaction and that ascorbic acid could be titrated with chloramine-T using, 2,6-diclhlorophenolilndophenolas indicator. This titrimetric finishing step is based on the method (17)of determining several oxidizing agents including chloramine-T by titrating with ascorbic acid using 2,6-dichlorophenolindophenol as indicator. 2,6-Mchlorophenolindophenolhas been used as the titrant for the determination of ascorbic acid (18). Moreover, 2,6-dichlorophenolindophenolhas been found to oxidize thiols (19,201 and the mechanism of this reaction has ‘Address correspondence:
1406, Napier Town,Jabalpur-
C 0 Skri B. P. Pandey, Advocate,
4
82 001, India.
also been explored in detail (21,22). Consequently, a mild oxidizing agent like 2,6-dichlorophenolindophenol (redox potential 4-0.217) or any other oxidizing agent could not be used for the determination of ascorbic acid in the presence of thiols. On a project on the determination of sulfur cornpounds (23, 24) it was found that tetrachlorobenzoquinone reacts stoichiometrically with ascorbic acid but does not react a t all with strong reducing agents like thiols under identical conditions. The present paper reports on a direct spectrophotometric method of determining ascorbic acid with tetrachlorobenzoquinone by measuring the absorbance of the reaction mixture at 336 nm against a reagent blank. Visual and potentiometric methods have also been developed for the titration of ascorbic acid in the presence of EDTA, as shown in Figure 1. EDTA acts as an indicator as well as a trapping agent for certain metal ion impurities which may generally be ascribed with ascorbic acid samples (25). The procedure developed has also been applied for the determination of vitamin C in pharmaceutical preparations (tablets and injections). Resolution of mixtures of vitamin C with thiols has been successfully carried out by f i s t titrating the vitamin C content with tetrachlorobenzoquinone till the orange-red color appears. Upon dilution of the contents, thiols can be titrated with standard chloramine-T solution. Thiols are quantitatively oxidized to their corresponding disulfides with chloramine-?’ in the presence of potassium iodide. CH8C6H4SO2NC1-Na+C 21- f 2H++ CH3C6H4SO2NH2 Cl-Na+ 2RSH
+ I2
-
+
RSSR
+ 2HI
+ I2
EXPERIMENTAL SECTION Reagents. Ascorbic acid solution, approximately 1 x lo-* M (0.01 M), was prepared and standardized against 0.1 M sodium hydroxide solution. This was prepared fresh daily. The analyzed tablets were purchased locally. Their solutions were also prepared immediately before use and 2 mL of the
0003-2700/82/0354-0793$01.25/0 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
794 I
Table I. SDectroDhotometric Determination of Ascorbic Acid with f'etracdlorobenzoquinone serial no.
1
2 3 4 5 6 7 8
9 a
amt of ascorbic acid, mg taken founda 15.25 13.44 11.20 10.50 9.80 8.80 7.925 7.052 6.164
std dev 0.0925 0.0865 0.0932 0.1025 0.0892 0.0925 0.1210 0.1312 0.1380
15.00 13.24 11.02 10.29 8.982 8.766 8.102 6.901 5.982
Average of three determinations.
sample solution contained about 5.86 mg of ascorbic acid (0.0166 M). Tetrachlorobenzoquinone (Chloranil) solution 0.01 M was prepared by dissolving 0.2518 g of the Fluka A.G. Switzerland sample in 100 mL of acetone. The solution was stored in a dark vessel and kept in a cool place. Ethylenediaminetetraacetic acid, 0.01 M, was prepared by dissolving 0.372 g of the sodium salt sample (B.D.H.) in 100 mL of doubly distilled water. Thiols were samples supplied by Evans Chemetics, New York. All the chemicals used were of analytical grade. Apparatus. The absorbances were measured with a Pye-Unicam SP 8-100 spectrophotometer with 1-cm glass cells. Potentials were measured with a Toshniwal potentiometer using a combined glass-calomel electrode assembly. Procedure. Determination of Vitamin C with tetrachlorobenzoquinone. Spectrophotometric Method. The Io of the spectrum was obtained by using acetone as a solvent in both the reference and sample cells. The solvent in the sample cell was replaced by a standard M. The solution of tetrachlorobenzoquinone, 7 X was found at 336 nm. The wavelength was varied and the ,A, E value, 535 cm2 mol-l, was calculated by using the relationship A = cC1. An aliquot containing 15.25-6.164 mequiv of ascorbic acid solution is added to a 50-mL Erlenmeyer flask. To this, an excess (7 X lo4 M) solution of tetrachlorobenzoquinone was added and the contents were allowed to stand 5 min. Then an aliquot containing 5 mL of the reaction mixtures was selected and was placed in a reference cell. The excess of the tetrachlorobenzoquinone was measured by observing the absorbance at 336 nm. The residual amount of the reagent was
determined by a Beer's law calculation. Visual Method. An aqueous aliquot containing 70.45-10.56 meqdv of ascorbic acid is added to an Erlenmeyer flask. To this was added 1 mL of a 0.01 M EDTA solution and the contents were titrated against standard tetrachlorobenzoquinone solution. Here EDTA acts as an indicator as well as a trapping agent for associated metal ion impurities. The end point was sharp, stable, and evident by a golden yellow color formed with the first drop in excess of the titrant. Potentiometric Method. An aqueous aliquot containing 52.84-17.64 mequiv of ascorbic acid was taken in a 100-mL beaker fitted with automatic stirrer. The contents were titrated against standard tetrachlorobenzoquinone by adding 0.1 mL of titrant and noting the potential change. The end point is indicated by a sufficiently large (70 mV) potential jump. Determination of Vitamin C in Pharmaceutical Preparations. A finely ground tablet was stirred with about 30 mL of doubly distilled water containing 1 niL of glacial acetic acid. Mter the mixture was allowed to stand for 15 min, the residual solid was fiitered on Whatmann No. 42 paper and washed with water. The filtrate was made up to 50 mL in a volumetric flask. Injections were directly diluted. An aliquot containing 5-10 mL of solution was taken in a 100 mL Erlenmeyer flask. It was then diluted with acetone (=lo mL) to avoid precipitation of the reagent. One of two milliliters of EDTA (0.01 rn) solution was added and titrated against 0.01 M standard tetrachlorobenzoquinone till a golden yellow color appeared to indicate the end point. Resolution of Mixtures of Vitamin C and Thiols with Tetrachlorobenzoquinone. An aliquot containing 15-50 mg of vitamin C was taken in a 100-mL Erlenmeyer flask. To this was added 10-30 mg of thiol sample, and the mixture was diluted with 5-10 mL of acetone to avoid precipitation of the reagent. Then 1mL of 0.01 M EDTA solution was added (to avoid the interference of metal ion impurities) and allowed to stand for 5 min. The contents were thoroughly shaken during this period and titrated with standard 0.01 M tetrachlorobenzoquinone solution, with constant shaking after each addition of the titrant. The end point is indicated by the appearance of a red-orange color formed from the first excess drop of the reagent with the thiol content. After the contents were diluted with 20 mL of water, 0.5 g of potassium iodide, 5 mL of 10% sulfuric acid, and 1mL of 1% starch were added and the thiol contents were subsequently titrated with 0.01 M standard chloramine-?' solution.
RESULTS AND DISCUSSION Table I summarizes the results of ascorbic acid determinations by the spectrophotometric method. The recoveries
Table 11. Titrimetric Determination of Ascorbic Acid with Tetrachlorobenzoquinone amt of ascorbic acid, mg ____ visual method serial no.
1
2 3 4 5 6 7
8 9 10 a
taken 70.45 61.64 52.83 44.00 35.22 26.41 20.93 17.67 14.00 10.56
founda
% error
70.27 61.78 52.71 44.08 35.10 26.29 20.97 17.71 13.94 10.59
-0.25 +0.22 -0.22 t0.18 -0.34 -0.45
Average of six determinations,
1.0.20
+0.23 -0.42 -1-0.28
std dev 0.0734 0.0424 0.0424 0.0542 0.0452 0.0834 0.0678 0.0764 0.0424 0.0770
comparison methodb 70.27 61.81 52.83 44.10 35.18 26.28 20.88 17.85 13.98 10.62
Bv iodometric method ( 2 6 ) .
--
__
potentiometric method
taken
foundc
% error
std de;'
52.84 44.03 35.22 26.00
52.66 42.79 35.22 25.89
-0.34 -0.54
0.0704 0.0942
-0.44
0.0835
17.61
17.10
+0.51
0.0905
io.00
Average of three determinations.
10.00
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
795
Table 111. Determination of Vitamin C in Pharmaceutical Preparations amt in mg sample sample
taken
founda
Suckcee (IDPL) tablet Cilin (Glaxo) tablet Chewcee (Lederle) tablet Cecon (Abott) tablet Redoxon (Roche) injection
500 500 500 500 500
503 491 505 502 493
no.
1
2 3 4 5 a
Average of six determinations.
% error t 0.60
-1.80 t 1.00 t0.40 -1.40
comparison methodb 505 492 503 504 4 94
% error
+ 1.oo -1.60
t 0.60
1-0.80 -1.20
By the iodometric method. I
Table IV. Determination of Mixtures of Vitamin C with Thiols mixascorbic acid in mg ture mixture taken founda % error no. 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 a
vi.tamin C + 1-propanet hiol vitamin C t 2-mercaptoethanol vitamin C t cystamine hydrochloride vitamin C + thiolactic acid
Average of four determinations.
17.61 20.93 35.22 44.03 15.85 19.37 29.94 51.00 17.65 22.90 33.46 44.34 15.85 20.93 36.98 47.55
17.45 21.10 35.65 44.40 16.00 19.60 30.12 50.50 17.85 23.10 33.20 44.80 15.66 20.65 37.14 47.10
0
OH- C - H I
CH20H
Tetrachlorobenzoquinone
o= n -
“J
-0.9803 t1.135 t 0.8733 -0.7170 t 0.7923 -1.198 -1.098 t 0.4326 -0.9463
-0.9829 t 1.144
-0.8284 -1.178 -0.7692 -0.6756 -0.8093 +1.000 -0.6641 -0.7448 -0.9398 t 1.037 -0.9823 -1.053 t0.9652 t1.068
ACKNOWLEDGMENT I thank Ashutosh Srivastava for valuable discussions.
C I
HO-
t 1.180 t 0.601 2
30.22 26.62 20.35 15.20 32.25 26.46 22.08 18.00 31.41 25.30 21.08 16.55 30.24 24.42 20.92 16.08
developed has been applied for determining the vitamin C content of pharmaceutical preparations and for resolving mixtures with thiols, the results of which are summarized in Tables I11 and IV. Mixtures of ascorbic acid with thiols, like cysteine, 0-mercaptobenzoic acid, mercaptosuccinic acid, and 3-mercaptopropionic acid could not be resolved by the proposed method. Although selective oxidation of ascorbic acid is feasible by tetrachlorobenzoquinone in preference to thiols, as stated, no reason could be found for this abnormal phenomenon. However, the method gives excellent results in other pharmaceutical preparations and mixtures, as listed.
d
4rcorb;c Ac;d
t 0.9463
30.52 !16.31 20.52 15.28 32.50 26.64 22.24 17.82 31.62 25.51 21.28 16.38 30.54 24.68 20.72 15.91
Standardization with chloramine-T (27).
HO-C
H - CI
-0.9085 t 0.8122 t 0.4310 t 0.8403
thiol content in mg taken foundb % error
-
C-H
OH
I
CHBOH
Dehydroarcorblc a c i d
fetrachlorohydroquinone
Figure 1.
are higher for the smaller amounts of sample; for 6 mg the standard deviation ranged to 0.1380. Results of visual and potentiometric methods are given in Table 11. The results are accurate in the range, h0.18 to f0.45%. EDTA acts as an indicator as well as trapping agent for other metal ion impurities also. Diphenylamine can also be used as an indicator but it needs a nonaqueous medium, otherwise further dilution is necessary to avoid precipitation of the reactants. Substances that do not interfere with either indicator when present in amounts up to 10-15 M excess of vitamin C include, glucose, sucrose, maltosle, citric acid, oxalic acid, tartaric acid, and amino acids like serine, glycine, and alanine. The method
LITERATURE CITED (1) Rao, G. G.; Sastrl, G. S. Anal. Chim. Acta 1971, 5 6 , 325. (2) Sastri, G. S.; Rao, G. G. Talanta 1972, 19, 212. (3) Paul, R. C.; Chauhan, R. K.; Prakash, R. Indian J . Chem. 1971, 9 , 879. (4) Eremlna, 2. I.; Gurevlch, V. G. Zh. Anal. Khlm. 1964, 19, 519. (5) Chandra, S.; Yadav, K. L. Microchem. J . 1968, 13, 586. (6) Murthy, C. N.; Bapat, N. G. Z . Anal. Chem. 1963, 199. 367. (7) Deshmukha, G. S.: Bapat, N. G. 2. Anal. Chem. 1955, 145, 254. (8) Leonhardt, H.; Moeser, W. Z . Anal. Chem. 1941, 122, 3. (9) Erdy, L.; Bodor, E. Anal. Chem. 1952, 2 4 , 418. (10) Rao, 0 . G.; Rao, V. N. 2.Anal. Chem. 1955, 147, 338. (11) Devanl, M. 6.; Shlshoo, C. J.; Patel. N. N. Ind. J . Pharm. 1967, 29, 98. (12) Bhattacharya, H.; Gangull, S. K. Indian J . Pharm. 1964, 26, 200. (13) Stevens, J. W. Ind. Eng. Chem., Anal. Ed. 1938, IO, 269. (14) White, V. R.; Frltrgerald, J. M. Anal. Chem. 1972, 44, 1267. (15) Gupta, D.; Sharma, P. D.: Gupta, Y. K. Talanta 1975, 22, 913. (16) Verma, K. K.; Gulatl, A. K. Anal. Chem. 1980, 52, 2336. (17) Svehla, G.; Kolal, L.; Erdy, L. Anal. Chim. Acta 1963, 2 9 , 442. (18) Erdy, L.; Svehla, G. Chemist-Analyst 1963, 52, 24. (19) Basford, R. E.; Heunnkens, F. M. J . Am. Chem. Soc. 1955, 7 7 , 3873. (20) Overberger, C. G.; Bonslgnore, P. V. J . Am. Chem. Soc. 1956. 80, 5431.
796
Anal. Chem. 1982, 5 4 , 796-799
(21) Pandey, N. K.; Mlshra, K. K. Indlan J . Chem., In press. (22) Pandey, N. K.; Mishra, K. K.; Kashyap, M. J. Phosphorus Sulfur, in press. (23) Srlvastava. A,: Bose. S. Tahnta 1877.. 2 4 . 517. i24j Srlvastava; A.'Talanta 1979, 20, 917. (25) Berka, A.; Vultarin, J.; Zyka, J. "Newer Redox Titrants"; Pergamon: Oxford, 1965; p 162. (26) Ballentine, R. Ind. Eng. Chem. Anal. Ed. 1941, 13, 89.
(27) Paul, R. C.; Sharma, S.K.; Kumar, N.; Kumar, S. Talanta 1875, 22(3), 311.
RECEIVED for review August 7,1981. Accepted November 17, 1981. Thanks are due to the University Grant Commission (New Delhi) for a research fellowship.
Resolution of Mixtures of Organic Acids by Conductometric Titrations in 2-Methoxyethanol Carlo Pretl, Lorenzo Tassi, and Giuseppe Tosl" Zstituto di Chimica Generale ed Znorganica, University of Modena, 4 1100 Modena, Italy
A study has been made on the conductometrlc tltratlons of mlxtures of organic aclds In 2-methoxyethanol as solvent and wlth 0.10 M N,N'-dlphenylguanldlne (DPG) as standard tltrant. Conductometrlc tltratlon curves are presented and discussed for both allphatlc and aromatlc mono- and dlcarboxyllc aclds and para- and meta-substltuted benreneselenlnlc aclds In binary, ternary, and quaternary mlxtures. The solvent is suitable for the resolutlon of acld mlxtures In some cases where a quantltatlve determlnatlon Is lmposslble In aqueous solutions owing to very close pK values.
In a preliminary study on the conductometric determination of acids in 2-methoxyethanol it was observed that dibasic acids, both aliphatic and aromatic, gave two end points a t a 1:l and 1:2 acid/base stoichiometry using N,N'-diphenylguanidine (DPG) as primary standard titrant (I). Furthermore, the triprotic citric acid (pK1 = 3.14, pK2 = 4.77, and pK3 = 6.39 at 18 "C in aqueous solution (2)) gave three end points corresponding to 1:1,1:2, and 1:3 acid/base ratio, the conductance behavior being linear beyond a theoretical 1:4 acid/base stoichiometry. This result indicates the possibility of performing differential titrations of acid mixtures in cases where such determinations should be impossible in aqueous solution owing to the very close pK values. The titrations reported in this paper were mainly done under the experimental conditions used in the previous study ( I ) and the present results pertain to these conditions only. In the same way we have restricted our study to titrations at room temperature although, as reported in the literature (3), in some cases higher temperatures might be favorable. EXPERIMENTAL SECTION The conductance measurements in 2-methoxyethanol were carried out with the same instrument as previously described ( I ) . The relative precision of the resistance readings was ca. 1.0%. The solvent, 2-methoxyethanol, supplied by Carlo Erba in high purity grade and containing 0.05% water (by Karl Fischer titration) was used without further purification; the absence of conducting impurities was tested for in many blank titrations before the study. N,N'-Diphenylguanidine (DPG), supplied by Fluka (purum 98%),was twice purified by recrystallization from hot toluene (mp 150 "C; lit. 150 "C ( 4 ) ) . All the acids, commercially available or prepared according to literature data, were reagent grade. The accurately weighed acid samples were mixed and dissolved in 50.0 mL of the solvent and then were titrated with lo-' M DPG 0003-2700/82/0354-0798$0 1.2510
in the same solvent from an automatic digital buret, AMEL Model 233. The titrant was added in l/lo-mL portions under magnetic stirring, and then with the stirrer off, the equilibrium reading of the bridge was taken after each addition; equilibrium was reached within a minute and the resistance readings were stable with time. We have never observed under the experimental conditions the formation of any precipitate. Volume corrections were applied to all the conductance data used.
RESULTS AND DISCUSSION The following mixtures of acids were titrated: (a) binary mixtures both of benzeneseleninic and of monocarboxylic acids; (b) binary mixtures of dicarboxylic and monocarboxylic acids; (c) ternary and quaternary mixtures of monocarboxylic acids. The analytical results and the titration curves of the acids investigated are reported in Tables I and I1 and in the Figures 1-5, respectively; all the results are the average of at least duplicate titrations. The curves are plotted in units of conductance vs. mL of base in all the titrations, applying volume corrections to all the data. Straight lines have been calculated by the least-squares method including almost all the data points, and they intersect at the theoretical integral base:acid ratios, as clearly shown in the figures. The conductance curves have been shifted vertically in Figures 1-3 to present the results more clearly. Binary Mixtures of Acids. Benzeneseleninic Acids. From the results it appears that resolution is possible for mixtures of benzeneseleninic acids of the type XC6H4Se02H (X = H, p-C1, m-C1, p-Br, m-Br, p-NO,, m-NO2,p-CHJ whose pK values range between 4.70 and 4.30 in water a t 25 OC (5). An example of the titration curves of a mixture of benzeneseleninic acid and 4-bromobenzeneseleninic acid is reported in Figure 1 and the analytical results are presented in Table I. It is worthy of note that the sequence of equivalence points is the same as expected in aqueous solution on the basis of the pK values. Monocarboxylic Acids. Figures 1and 2 show some representative titration curves of monocarboxylic acid mixtures. The end points can be determined accurately, yielding satisfactory recoveries (Table I). The first end point corresponds generally to the neutralization of the strongest acid in the mixture, and the second end point to the weakest acid, referring to their strength in water. However, the above sequence is frequently reversed when the aqueous pK values of the acids in the mixture are similar (see, for example, the titrations of 4-hydroxybenzoic and 4-methoxybenzoic acids 0 1982 American Chemical Society
