The apparent transmittance of the solution is ---=T(-) Peolution Tapparent Psolvent
+ F(T) + F(1)
AT T 1 , Equation 3 may be rewritten as
Since Aapparent- A (Tspyent
1 1
)
AA
=
=
AA
-0.434
=
(3)
=
-0.434
0.434 (F!%,”f’)
and
( : =a
0434 F(1) - F(T) A F(1) 1
+
)
(4)
The calculation of the relative absorbance error has been carried out for aqueous solutions at 25 “ C in quartz cells and the results are tabulated in Table I. It has been assumed for the calculation that the refractive index of the absorbing solution does not differ appreciably from that of pure water.
Values given in Table I were calculated with F(T) including terms for the thirty-one rays which undergo four reflections. However, if only the six doubly-reflected rays are considered, the calculated 6’s differ from those given in Table I by less than 0.5%. It is apparent then, that F(T) converges quite rapidly, and no practical advantage is gained by including more than the first six terms. In summary, all conventional absorbance measurements include a systematic bias due to multiple reflections within the cell. The magnitude of this bias is easily and accurately calculated from the refractive indices of the solution and the cell at the wavelength of interest. The appropriate refractive indices are used in Equation 1 to calculate reflection coefficients, which are substituted in Equation 2 along with the solution transmittance. Equation 4 may then be evaluated to find the relative absorbance error. The error commonly amounts to 1 to 2 parts per thousand for aqueous solutions and will be slightly smaller for most organic solvents. RECEIVED for review June 12, 1972. Accepted September 15, 1972.
Mercuric Iodate as an Analytical Reagent. Spectrophotometric Determination of Certain Anions by an Amplification Procedure Employing the Linear Starch Iodine System Willie L. Hinze and Ray E. Humphrey Department of Chemistry, Sam Houston State University, Huntsville, Texas 77340 SEVERAL RELATIVELY INSOLUBLE metal iodates have been used for the determination of anions which form a less soluble or undissociated compound with the metal ion involved. The anion being measured is “traded” for the iodate ion which is released into the solution. Iodide ion and acid are added to yield triiodide ion. These reactions are shown in the equations below for the simplest case.
+ X- MX + + 81- + 6H+ 313- + 3H10
MI03 103-
+
103-
(1)
spectrophotometrically employing a metal iodate. Silver iodate was used and the absorption of the triiodide (6, 7) or the starch iodine complex measured (7, 8). In this work, the absorption of the starch-iodine complex was employed for the spectrophotometric determination of bromide, chloride, cyanide, iodide, sulfide, sulfite? thiocyanate, and thiosulfate. These methods are about as sensitive as any which have been reported for most of these anions and have higher sensitivity for some.
+
The triiodide formed is then either titrated with standard sodium thiosulfate solution, the absorption determined, or the starch-iodine complex measured spectrophotometrically. The procedure has been called an “amplification” reaction when a titration is involved as 6 iodine atoms are produced for each monovalent anion exchanged ( I ) . Chloride has been determined titrimetrically in this way using either mercuric or mercurous iodate ( I ) , or silver iodate ( I , 2 ) , fluoride has been determined with calcium iodate (3) while barium iodate has been applied to the measurement of sulfate (4, 5). Apparently chloride is the only anion which has been determined (1) R. Belcher and R. Goulden, Mikrochim. Acta, 1953,290.
(2) J. Sendroy. Jr., J . Biol. Cliem., 120,405 (1937). (3) W. I. Awad, S. S. M. Hassan, and M. B. Elayes, Mikrochim. Acta, 1969,688. (4) D. A Webb, J . Exp. Biol., 16,438 (1939). (5) J. L. Lambert and D. J. Manzo, Anal. Cl7im. Acta, 54, 530 (1971).
EXPERIMENTAL
Apparatus. Absorption measurements were made with Beckman ACTA 111, DB-G, and DK-2A spectrophotometers. An International clinical centrifuge model CL and International Model SBV Centrifuge were used to separate the mercuric iodate from the solutions. Reagents. Mercuric iodate was obtained from City Chemical Corp., New York, N.Y. A mortar and pestle was used to reduce the chemical to a fine powder. Compounds used to provide the anions studied were Baker and Adarnson or Baker analyzed reagent grade materials. The starch iodide reagent was prepared using Baker analyzed cadmium iodide and Fisher Scientific Co. potato starch. Stock solutions of the various anions were in the range of 0.01 to 0.001M using 1 :1 ethanol-water as solvent. Sulfite solutions contained 5 glycerol to retard oxidation. (6) F. L. Rodkey and J. Sendroy. Jr., Clii7. Clienz., 9, 668 (1963). (7) J. Sendroy, Jr., J . Biol. Chem., 120,419 (1937). 27,444 (1955). (8) J. L. Lambert and S. Y . Yasuda, ANAL.CHEM.,
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
385
Table I. Effective Molar Absorptivities and Useful Concentration Ranges for Anions Effective Dilumolar Range, Anion tiona absorptivityb ppm BrHigh 4,700~ 2.8-28 20,000 0.5-7.5 Low 4,600 0.7-15 cIHigh O.2-3.5 Low 20,000 4,600 0.8-10 CNHigh Low 20,000 0.1-2.6 IHigh 4,600 3.8-40 O.5-10 Low 22,000 5,200 1 .4-20 SCNHigh Low 20,000 0.3-5.0 S 2High 8,200 0.4-8.5 Low 40,000 0.1-1.6 so32High 9,500 1.OM7 Low 40,000 0.2-4.2 szo32High 9,000 1.5-20 Low 43,000 0.3-5.5 High dilution was 5 ml to 100 ml. Low dilution was 24 ml to
a t 595 nm after allowing the color t o develop for I O minutes. This is the low dilution procedure. For the higher concentration range, a measured volume of the anion solution was put in a n Erlenmeyer flask and diluted to 10 ml with 1 :1 ethanol-water. Mercuric iodate, 30-40 mg, was added and mixed thoroughly. After 15-30 minutes reaction time, the solutions were centrifuged for 5 minutes in a clinical centrifuge. A 5-ml aliquot was transferred to a volumetric flask containing 85 ml of distilled water and 2 ml of 2.5M HCI. Three milliliters of the starch iodide reagent were then added and the solutions diluted to 100 nil with water. Absorbance values were measured a t 625 nm after 10 minutes. This is the high dilution procedure.
RESULTS AND DISCUSSION
The linear starch iodide reagent was prepared according to the procedure of Lambert and Zitomer (9). A cadmium iodide solution was prepared by dissolving 11 grams of CdI? in 450 ml of distilled water, boiling to remove any traces of iodine, and diluting to 500 nll. This solution was then brought to a boil and 20 grams of potato starch were added. After boiling and stirring for 20 minutes, the solution was centrifuged for 5 minutes, transferred to an amber bottle, and diluted to 1 liter. This reagent was stable for 2 to 3 months. Procedure. For the lowest concentration range, a measured volume of the anion solution was put in a 25-ml flask and diluted to 24 ml with 1 :1 ethanol-water. Approximately 25-35 mg of mercuric iodate was added and the flasks were shaken vigorously for about 1 minute until the solutions appeared “cloudy .” Thorough mixing is extremely iniportant. After the solutions had been allowed t o stand for 15-30 minutes with occasional shaking, 1 ml of 0.025M HNOa in 1 :1 ethanol-water was added. The acid lowered the blank apparently by decreasing the solubility of the iodate ( I ) . After a few minutes, the solutions were centrifuged for 5 minutes a t 3500 rpm and then filtered through Whatman No. I filter paper. Essentially complete separation is very important. A graduated pipet was then used to transfer 24 ml to a volumetric flask containing 70 ml of distilled water and 2.5 ml of a 2.5M HCI solution. Next, 3 ml of’ the linear starch iodide reagent were added and the solutions diluted to 100 ml with distilled water. Absorbance values were measured
Absorption of the Starch Triiodide Complex. The familiar starch iodine “blue” species show rather broad absorption with a maximum in the range of 580-630 nm depending on the amount of ethanol present. As the amount of ethanol is increased, the intensity of absorption decreases and the maximum shifts to shorter wavelengths. In water only, the maximum absorption is a t 630 nm with a molar absorptivity based on the concentration of Izof 40,000. In a solution containing 25 %, by volume, of ethanol the maximum is a t 565 nm and the molar absorptivity has decreased to about 23,000. The starch iodine complex does not form with 50% ethanol present. In the procedures used in this work, approximately 12 % ethanol was present in using the lower dilutions for lower concentrations and 2.5 for the greater dilutions and higher levels. Absorbance values were measured a t 595 nm for the low concentrations and a t 625 nm for higher amounts. Beer’s law was obeyed by iodate ion using the two procedures involving different dilutions with different amounts of ethanol in the final solution. The molar absorptivity based on the iodate concentration in the final solution was 92,000 for the 24-100 dilution and 110,000 for the 5-100 dilution. Iodate ion can be determined over the range of 0.2 to 3.5 PPm. The starch iodine “blue” species is formed quite rapidly and is remarkably stable. Absorbance values were read after ten minutes or somewhat longer and, in most instances, did not change significantly for hours and even days. Temperature changes are known to affect the intensity of absorption (10). We found the intensity to be considerably higher a t 10” than a t 50 “C. For example, with 2.5 ppm IO3-, A = 1.7 at 10 “C and 1.2 at 50 “C. Sensitivity of the Methods. The rather high molar absorptivity of the starch-iodine chromogen combined with the chemical “amplification” involved in the reaction result in spectrophotometric methods useful for the measurement of the anions involved in the very low parts per million range. The high absorptivity of the chromogen makes it necessary to use a dilution procedure and a mixed alcohol-water solvent to decrease the solubility of mercuric iodate. Effective molar absorptivities for the anions investigated in this study for the two dilution procedures are shown in Table I. As is evident, values for the divalent ions are approximately equal and are twice as large as the values for the monovalent ions, also about equal. The divalent anions yield two iodate ions while the exchange is 1: 1 for the monovalent anions. An amplification is involved in each case since each iodate on being reduced with iodide produces three triiodide ions. Determination of Chloride, Cyanide, Bromide, Iodide, and Thiocyanate. Mercuric ion reacts with these anions to form
(9) J. L.Lambert and F. Zitomer, ANAL.CHEM., 35,405(1963).
(10) J. L. Lambert, ANAL.CHEM., 23, 1247 (1951).
100 ml. For the high dilution, absorbance measured at 625 nm. For the low dilution, absorbance measured at 595 nm. Molar absorptivity values are based on the concentration of the
anion before the final dilution.
Table 11. Recovery Data for Chloride and Cyanide C1-, ppma CN-, ppm“ Error,
Error,
Present Found 0.45 0.89 1.78 2.67
z
Present
Found
2
-6.6 0.18 0.19 $5.5 -7.8 0.35 0.37 +5.7 +6.2 1.40 1.48 +5.7 -4.9 2.18 2.20 + 1 .o a Low dilution procedure (24-loo), A measured at 625 nm.
386
0.42 0.82 1.89 2.59
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
a very slightly dissociated compound. Effective molar absorptivity values for the singly charged anions are approximately the same, E = 20,000 for the low dilution procedure and E = 4,600 for the high dilution. The concentration levels which can be measured are in the range from less than 0.2 ppm to as high as 40 ppm depending on the dilution and anion sought. Concentration levels for the various anions are presented in Table I. The methods described might be of most importance for the determination of chloride and cyanide ions, since analysis for these anions is probably more common. Recovery data indicating the precision of the methods are presented in Table 11. Beer’s law is followed closely for both dilution procedures and precision appears to be acceptable for such absorption measurements. The highest effective molar absorptivity for chloride using the lowest dilution factor allows the determination for the anion at very low levels with reasonable accuracy. The limit on sensitivity is the blank due to the solubility of the mercuric iodate which makes a dilution necessary. On trying to increase the sensitivity by using a lower dilution, difficulty arises due to the increase in the blank and the decrease in absorption of the chromogen with greater amount of ethanol. The dilutions factor used, 24-100, probably represents a good compromise between these opposing effects. The molar absorptivity based on the chloride concentration appears to be about 4-5 times higher than the widely used methods employing mercuric thiocyanate and measuring the absorption of the FeSCN2’ species. The method described here is also considerably more sensitive than the mercuric chloranilate procedure measuring either the visible or ultraviolet absorption of the chloranilate ion (11, 12). Mercuric iodate seems to be better than silver iodate for determining chloride by use of the starch-iodine species as it is less soluble and a lower dilution can be employed. The effective molar absorptivity for chloride using silver iodate involving a dilution of approximately 1-250 is estimated to be about 350 using data from the literature (8). The earliest report on the use of silver iodate and the starch iodine complex was concerned with analysis of biological fluid samples for chloride ( 7 ) . The high stability of the starch iodine chromogen and resulting stable absorbance readings are due to the “linear” cadmium iodide-starch reagent developed by Lambert (13). The absorption of the triiodide ion by itself is also intense and has been used for determining chloride ion (6, 7 ) . Results for cyanide ion are approximately the same as for chloride, as is evident from the data in Table 11. The molar absorptivity based on cyanide for the Konig procedure, which is probably the most common colorimetric method for cyanide, is estimated to be about 25,000 from literature data (14). The high effective molar absorptivity for cyanide of the mercuric iodate method allows the measurement of this ion down to less than 0.2 ppm. There do not appear to be any reports in the literature of the application of such an amplification reaction to the determination of cyanide either titrimetrically or spectrophotometrically although reference to unpublished work apparently involving thiosulfate titration of iodine was found (15). Molar absorptivities, Beer’s law data, and recovery data for other monovalent anions, bromide, iodide, and thio-
(11) J. E. Barney I1 and R. J. Bertolacini, ANAL.CHEM.,29, 1187 (1957). (12) R.J. Bertolacini and J. E. Barney 11, ibid., 30,202(1958). (13) J. L.Lambert, ibid., 23, 1247 (1951). (14) L.S. Bark and H. G. Higson, Taiania,11,621 (1964). (15) H. Weisz, Mikrochirn. A m , 1970,1057,
Table 111. Recovery Data for Sulfide and Sulfite S 0 5 , ppm=
S2-, ppma
Error, Present
Found
z l
~-
Error, Present
Found
7 0
0.32 0.32 0 0.41 0.40 -2.4 0.63 0.64 +1.2 1.25 1.36 +8.8 0.79 0.82 +3.8 2.08 2.00 -3.8 1.58 -3.8 4.16 1.52 3.82 -8.1 a Low dilution procedure (24-loo),A measured at 625 nm.
cyanate compare very well with values for chloride and cyanide. Since these anions are probably of lesser interest, detailed data ( n their measurement are not included. Determination of Sulfide, Sulfite, and Thiosulfate. Two iodate ions are released for each of these anions so that corresponding effective molar absorptivity values are essentially twice as large as those for the singly charged ions (Table I). Beer’s law is followed very well by these anions. Precision for measurement of sulfide and sulfite is satisfactory as indicated by the recovery data in Table 111. The determination of sulfide and sulfite presumably is of considerable importance in certain pollution studies while thiosulfate is of lesser interest so that data are not included for that anion. This amplification method for sulfide is possibly more sensitive than the methylene blue procedure which has a molar absorptivity of approximately 35,000 (16). The sensitivity is about the same as for the reaction involving the displacement of the thiol anion of certain organic disulfides and measuring the ultraviolet absorption (17). The mercuric iodate procedure for sulfite is probably comparable in sensitivity in terms of effective molar absorptivity to the West-Gaeke method which has a molar absorptivity in the range of 37,000 to 48,000 (18). The iodate reaction is somewhat more sensitive than the method involving organic disulfides (19). The nature of the reaction between mercuric iodate and sulfite ion seems to be somewhat uncertain. Mercuric ion could be reduced by sulfite ion or these ions could interact to form a very stable compound or complex. Our evidence indicates that reduction of mercuric ion occurs (20). Other mercuric compounds, relatively insoluble mercuric chloranilate (21) and reasonably soluble mercuric thiocyanate (20), can be used to determine sulfite ion spectrophotometrically. There seem to be no literature reports on the spectrophotometric determination of either sulfide or sulfite by an amplification reaction. Silver iodide has been used for the titrimetric determination of sulfide by a procedure involving oxidation of the iodide released to iodate followed by reduction with iodide to iodine (22). Apparently no amplification reactions have been applied to the determination of sulfite titrimetrically. Possible Interferences. Since so many anions form very insoluble or undissociated compounds with mercuric ion, each (16) G. D. Patterson in “Colorimetric Determination of NonMetals,” D. F. Boltz, Ed., lnterscience, New York, N.Y., 1958, pp 273-1. (17) R. E.Humphrey, W. L. Hinze, and W. M. Jenkines 111, ANAL. CHEM., 43,140 (1971). (1 8) F.P. Scaringelli, B. E. Saltzman, and S. A.Frey, ibid., 39, 1709 (1967). (19) R. E.Humphrey, M. H. Ward, and W. L. Hinze, ibid., 42,698 (1970). (20) W.L.Hinze, J. E. Elliott, and R. E. Humphrey, ibid., 44,1511 (1972). (21) R.E.Humphrey and W. L. Hinze, ibid., 43, 1100 (1971). (22) H. Weisz and V. Fritsche, Mikrochim. Acta. 1970,638.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
387
anion is a potential interference for the others. If more than one of these anions is present in a sample, some type of separation would have to be devised. Oxidizing substances present would lead to high results by oxidation of the iodide added while reducing agents would cause low results by reacting with some of the iodine formed. However, cadmium iodide, which is used in the linear starch reagent, is resistant to oxidation (13). However, for those samples where there are no potential interferences, the mercuric iodate method should be advantageous. General Aspects of the Methods. The extent of reaction between all of these anions and mercuric iodate is quite high as can be seen by comparing the molar absorptivity values for iodate ion and a specific anion, corrected for the dilution factor. The reactions are in the range of 75-80z complete in 10-15 minutes. It is necessary to ensure good mixing to have the reaction be as complete as possible in minimum time. Separation of the excess solid from the solution prior to developing the color is of extreme importance. Filtration and centrifugation were both tried. Mercuric iodate seems to be somewhat easier to remove than mercuric chloranilate. Centrifugation is somewhat easier and faster and usually is sufficient. Results were possibly somewhat better for the lower levels when both separation methods were used. Different concentration levels for these anions could be determined by simply using different dilution factors. Effective molar absorptivities and concentration ranges are reported for 5-100 and 24-100 dilutions. In one trial, a 2-100
dilution was used and Beer’s law followed for chloride ion from 2-36 ppm, the effective molar absorptivity being 1800. Another approach to the problems of a high blank, other than a dilution procedure, is to add a constant amount of a sodium thiosulfate solution to each sample solution to remove an amount of iodine equivalent to that in the blank. This procedure was used in the method employing silver iodate (6). This was tried in one series of measurement for sulfite ion in which water was used as solvent and a 5-100 dilution used. The effective molar absorptivity for sulfite was 4800, Beer’s law being followed over the range of about 2-30 ppm sulfite. If interfering anions are not present or can be removed, this method for the determination of anions should be useful for accurate measurements at very low levels. The procedure is simple, reasonably fast, and requires a minimum of chemicals and manipulation. Reagents used are stable and impurities are not troublesome, as is sometimes the case with mercuric chloranilate (21). The color formed is also stable for a matter of hours. The possibility of adjusting the sensitivity by using different dilution factors should also be advantageous.
RECEIVED for review June 23, 1972. Accepted September 11, 1972. The authors express their appreciation to the Robert A. Welch Foundation of Houston, Texas for support of this Research. This work was part of the M.A. thesis of Willie L. Hinze, Sam Houston State University, May 1972.
Ethchlorvynol Reagent for Functional Groups Detection Frank F. Fiorese, Joerg N. Pirl, Paul R. Manio, and Anthony Carella’ Department of Public Health, Bureau of Toxicology, 1800 West Fillmore Street, Chicago, Ill. 60612
ETHCHLORVYNOL (l-chloro-3-ethyl-l-penten-4-yn-3-ol) is a hypnotic drug marketed under several trade names the most common being Placidyl. Several procedures for its detection have already been reported (1-4); Wallace and Washburn (2,5) pointed out that in the presence of strong acids such as HCI, the carbonyl derivative of ethchlorvynol, 1-pentyne-3one, is formed. This property could be appropriately utilized for analytical purposes as we found in this laboratory that in the presence of concentrated orthophosphoric acid, ethchlorvynol adsorbed to Florisil, will react with carbamates, primary aromatic amines, hydrazine, indole derivatives, and some sulfur-containing compounds to form different colored products. In this communication, we report data gathered with respect to the interactions of ethchlorvynol with representative classes of various chemical compounds.
Table I. Results of Interaction between Different Functional Groups and Ethchlorvynol Reactive Color Color in Color Chemical functional in CHC1, on in family group CHC13 standing EtOH Green Dark Discolors Carbamates //O R-CH,-C--SH: green Primary Red Yellow Dark aromatic yellow amines
1 Present address, Office of the Chief Medical Examiner, 520 First Avenue, New York, N.Y.
EXPERIMENTAL (1) C. J. Umberger, Office of the Chief Medical Examiner, 520 First Ave., New York, N.Y., personal communication, 1963. (2) J. F. Wallace, W. T. Wilson, and E. Dahal, J. Forensic Sci., 9, 342 (1964). (3) . , E. J. Alaeri, L. G. Kostas. and M. A. Luonogo, - . Amer. J . C1k. Parhof., 38, 125 (1962). (4) A. W. Davidson, J. Ass. Ofic. Aizal. Chem., 53, 834 (1970). (5) W. H. Washburn and F. A. Scheske, ANAL.CHEM.,29, 346 (1957). 388
Apparatus. All reactions were carried out on a 7 X 0.4 cm cylindrical glass tubing plugged at one end with glass wool and filled two-thirds with activated Florisil (60-100 mesh). Reagents. All reagents used were of analytical grade: 2 g per cent ethchlorvynol in ACS chloroform (ethchlorvynol reagent); concentrated orthophosphoric acid (OPA) and 95 ethanol.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
