Table V. Apparent Deviations from the Order of Replacement Replacement occups m Replacmg Con p k x
Se(DDC), Sb(DDC)? Bi(DDC), In(DDC)?
H2S04
HC104
Hg2+ Bi"
yes yes
yes yes
Sb" As +
no
no
no
no
Lon
I + HC1
0.11HC1
no
no no
partiallv
yes
no
from the K,, values given by Stary ( 1 8 ) (log K I , = 10.34, log K C d = 5.41). It was not always possible to represent these data by straight lines through the origin. This was almost certainly due to the fact that for some pairs equilibrium was not completely attained even though prolonged extraction times were used. However, it can be said that the difference in the l f n log K,, values are of the order of 0.1 for In/As and 0.3 for Mo/As and for TefSb. The established order of extraction should also be the inverse order of replacement. That is, a given metal A in the aqueous phase, when shaken with an organic solution of BDDC, should replace B from its complex if A stands on the left hand side of B in the order of extraction. This was checked by separate experiments and shown to hold. Some notable exceptions, specified in Table V, were caused by complexation either of A or B in HCl, others were caused by vanishingly slow reaction rates. A good example for inertness is Se(DDC)4 which, in HC104 or H2S04, can be replaced by Hg2+, but not as expected by Bi3+, Cu2+, or Te4+. Analytical Applications. A necessary condition for quantitative determinations by substoichiometric extraction as originally proposed by Stary ( 7 ) is the extraction of equal quantities of metal by equal quantities of reagent. It can be seen from the present work that in the systems studied (H2S04,HC104, or HCl), no difficulties arise for the extraction of Cu2+, Mo6+, In3+, Cd+*, and Pb+' with Zn(DDC)Z. However, the above condition cannot be met with metals forming mixed C1-DDC complexes without
stringent control of the C1 content. Therefore, in the case of Hg2+, As3+, Sb", and Te4+, substoichiometric extraction does not seem very useful in practical analytical work. A more attractive application is the complete extraction of one or several elements from acid solutions by the use of suprastoichiometric quantities of MeDDC as reagent, where Me is a metal standing immediately to the right of the sought element(s) in the order of extraction. This mode of operation can yield very pure fractions with only one extraction and without the need to add masking agents or to adjust the pH. Some work along these lines was recently published for three ions ( 1 9 ) ,but lack of knowledge of the order of extraction prevented a more general application. It should also be remembered that applications to systems with many cations will entail replacement reactions, some of which are slow to occur. Finally, it will be possible in special cases (as with Se4+ and Te4+) to take advantage of the inertness of the DDC complex in order to arrive at specific separations. LITERATURE C I T E D G. Eckert, Fresenius' Z. Anal. Cbem., 155, 23 (1957). H. Forster, J. Radioanal. Cbem., 4, l(1970). H. Bode and F. Neumann. Fresenius' Z. Anal. Cbem., 172, 1 (1960). G. D. Thorn and R. A. Ludwig, "The Dithiocarbamates and Related Compounds," Elsevier, New York, N.Y., 1962. A. Hulanicki. Talanta, 14, 1371 (1967). D. Coucouvanis, Progr. lnorg. Chem., 11, 233 (1970). J. Ruzicka and J. Stary. "Substoichiometry in Radiochemical Analysis," Pergamon Press, Elmsford, N.Y., 1968. J. Stary and J. Ruzicka, Talanta, 18, 1 (1971). N. Suzuki, Jap. Analyst, 21, 532 (1972). A. Elek eta/.,J. Radioanal. Cbem., 4, 281 (1970). J. Joris eta/.,Anal. Cbem., 41, 1441 (1969). G. Goldstein, "Equilibrium Distribution of Metal-ion Complexes," ORNL3620, USAEC, 1964. G. B. Briscoe and S. Humphries, Talanta, 16, 1403 (1969). F. Kukula eta/., J. Radioanal. Chem., 3, 43 (1969). A. Wyttenbach and S. Bajo, Helv. Cbim. Acta, 56, 1198 (1973). C. K. Jprgensen, J. lnorg. Nucl. Cbem., 24, 1571 (1962). G. St. Nikolov eta/.,J. horg. Nucl. Cbem., 33, 1059 (1971). J. Stary and K. Kratzer. Anal. Cbim. Acta, 40, 93 (1968). A. Elek, J. Radioanal. Chem., 16, 165 (1973).
RECEIVEDfor review April 9, 1974. Accepted August 16, 1974.
Fluorometric Assay for 5-Hydroxytryptophan with Sensitivity in the Picomole Range K. H. Tachiki and M. H. Aprison Section of Neurobiology, The Institute of Psychiatric Research and Departments of Biochemistry and Psychiatry, Indiana University Medical Center, Indianapolis, Ind. 46202
Optimizing conditions for the reaction of 5-hydroxytryptoPhan (5-HTP) with o-phthaldialdehyde has provided a sensitive method for the fluorometric assay of 5-HTP isolated from brain tissue. Samples from tissue containing as little as 20 Pmol of 5-HTP gave fhorescent values which are greater than twice blank values, whereas in the case of standards, levels as low as 3 pmol can be assayed. The increased sensitivity has made it possible to measure the endogenous levels of 5-HTP in small areas of the brain of the rat.
The compound n- phthaldialdehyde (OPT) is known to react with various compounds to form colored and/or fluorescent products ( 1 - 5 ) . The reactivity of OPT to a given compound is dependent on the conditions of the reaction such as temDerature. acidicitv or basicitv. concentration of OPT, and reaction time. Mailkel and Mrller ( 5 ) found that by employing acidic conditions, the OPT reagent had a degree of specificity for 3,5-substituted derivatives of indole compounds. Using this fact, they published a fluorometric ANALYTiCAL CHEMISTRY, VOL. 47, NO. 1, JANUARY 1975
7
I
3.0 I
0.02
0.04
0.05
0.08
0.1
0-PHTHALDIALDEHYDE (mg)
Figure 1. Determination of the minimal amount of OPT for fluorophor formation with D.L-~-HTP The amount of 5-HTP used in these studies was 500 pmol, the reaction temperature was 83 O C , the incubation time was 15 minutes, and the final normality of HCI was 6.7N. The fluorescence was determined at a wavelength of 480 nm using Corning glass filter No. 3850
method for assaying 5-hydroxytryptamine (5-HT). This method was reported t o be 20 times more sensitive than any method which relies on the native fluorescence of 5-HT in 3 N HCl. Since 1966, a number of publications (6-9) have appeared describing optimal conditions for the reaction of 5-HT with OPT. Comparable data are not available in the literature for other 5-hydroxyindole compounds even though descriptions of various experimental assay conditions for 5-HIAA and 5-HTP have been reported (see 1014). Our interest in the functional role of 5-HT and its immediate precursor 5-hydroxytryptophan (5-HTP) has necessitated the development of a method of assay which is sensitive enough to measure the endogenous levels of 5-HTP in specific areas of the brain. I t was felt that the use of the O P T reagent offered the most promise for this purpose. The data in this paper establish the optimal conditions ( i e . , reaction time, temperature, reagent corcentrations, etc.) for the reaction of O P T with 5-HTP. These data include identification of factors which affect the fluorescent yield of the 5-HTP derivative formed. EXPERIMENTAL Materials. Chemicals and reagents used were analytical grade HCI (Mallinckrodt Chemical Works, Pittsburgh, Pa.); o - phthaldiD,L-kynurinine, L-kynurinaldehyde, D,L-5-methoxytryptophan, ine, kynurenic acid, 5-hydroxytryptamine, ~ , ~ - 5 - h y d r o x y t r y p t o phan, and 5-hydroxyindoleacetic acid (Sigma Chemical Co., St. Louis, Mo.); L-5-hydroxytryptophan (Calbiochem, La Jolla, Calif.); nicotinamide and nicotinic acid (Nutritional Biochemical Corp., Cleveland, Ohio); anthranilic acid (Eastman Organic Chemicals, Rochester, N.Y.); 5-methyl-D,L-tryptophan (Mann Research Laboratories, Inc., New York, N.Y.); and reagent grade ethanol (Commercial Solvents Corp., Terre Haute, Ind.). Male rats (200-250 g) of Sprague-Dawley strain were obtained from Laboratory Supplies, Indianapolis, Ind. Methods. Fluorometric Analyses. All fluorometric studies were carried out using a recording Farrand spectrofluorometer. Flatbottomed tubes made from glass tubing (5-mm, 0.d.; 3-mm i.d. X 50 mm in length) served as cuvettes. Unless stated otherwise, a Corning filter No. 5840 was used between the light source and the first monochromator and a No. 3387 filter was placed in between the sample and the second monochromator. The first monochromator was set at a wavelength of 360 nm for excitation and the second monochromator was set to measure the fluorescent yield at 480 nm. All spectrofluorometric work was done a t room temperature. Preparation of Samples All standards were usually prepared in 0.01N HC1. The 5-HTP from brain tissue was isolated by a procedure described in detail elsewhere ( 1 5 ) using ion exchange chro8
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1 , JANUARY 1975
0
2.5
5 .O
I .5
10s
H C l ( F i n a l Normality)
Figure 2. Effect of the concentration of HCI on the OPT condensation reaction with L-5-HTP The fluorescence at 480 nm (uncorrected) was determined in a Farrand spectrofluorometer using an excitation wavelength of 360 nm (uncorrected). Each point represents the means f std dev of 3 determinations and t h e deviations of each point are marked by brackets or are within the area of the symbol matographic techniques. The HCl used in these studies was first tested for oxidizing impurities by the method described by Atack and Lindqvist (11 1. For measurement of the excitation and fluorescence spectra of standard L - ~ - H T P the following procedure was used. For fluorophor formation, 100 pl of 1 2 N HCl containing 0.06 mg of O P T were added to 50 pl of sample containing 20 pmol of L - ~ - H T in P a cuvette, the mixture was buzzed immediately, the top of the cuvette was covered with parafilm, a tiny hole was made with a needle, and the sample was placed in a 40 "C water both for 60 minutes. At the end of the incubation time, the cuvette was removed from the water bath, cooled with cold running tap water, and carefully dried. The sample was buzzed again and then the cuvette was placed in the Farrand spectrofluorometer. The excitation spectrum was obtained by measuring the emission at 480 nm whereas the fluorescence spectrum was determined using an excitation wavelength of 360 nm. No filters were used except in the case of the fluorescence spectrum where Corning glass filter No. 5840 was placed between the light source and first monochromator to eliminate light scatter effects.
RESULTS Typical excitation and fluorescence spectra of the fluorescent derivative formed by the condensation of O P T with 20 pmol of D , L - ~ - H Tindicated P an excitation maximum a t 360 nm (uncorrected) and a fluorescence maximum a t 480 nm (uncorrected). Optimum Concentration of OPT. At least 0.06 mg of OPT per 150 1 1 of final volume of sample are needed if the OPT is not to be a limiting component in the assay mixture (Figure 1). The time course of fluorophor formation with these same concentrations (see Figure 1) of OPT were also determined; these results support the conclusions drawn from the data in Figure 1. In addition, a t maximum sample fluorescence, the reagent blank readings with 0.08 mg of OPT were greater than twice that obtained with 0.06 mg of OPT. Therefore, unless otherwise stated, all subsequent studies were conducted using 0.06 mg of OPT per 150 1 1 of final volume. Optimum HCl Concentration. T o test the effect of the final normality of HC1 on fluorophor formation, 100 pmol of L - ~ - H T were P reacted for 60 minutes a t 40 "C with 0.06 mg of O P T in varying concentrations of HCl (Figure 2). A concentration of HCl of about 8N is needed for optimum fluorescence. Additional studies showed that another acid
0.8
5-HTP
/
5-HT
i
such as H2S04 could not replace HCl. Although the fluorescent yield increased with increasing normality of H2SO4 up to about 8N, the maximum fluorescence observed for 100 pmol of 5-HTP reacted in 8N HpSO4 was less than 30% of that observed with HC1. Optimum Heating Conditions. The fluorescent yield after reacting 200 pmol of 5-HTP with 0.06 mg of O P T increased by 12% as the temperature was raised from 40 to 60 "C and did not change thereafter up to 80 "C. However, an increase in fluorescence of the reagent blank was observed with the temperature change from 40 to 80 "C which more than offset the gain in fluorescence of the 5-HTP, particularly a t 80 "C. At 40 "C, the optimum time of heating for 5-HTP, 5-HT, and 5-HIAA was 60 minutes (Figure 3). The fluorescence for 5-HTP did not increase any further between l to 2 hr of heating a t 40 "C. The data also show an extremely large difference in reactivity of 5-HT and 5HIAA with OPT a t 40 and 60 "C (Figures 3 and 4). However, the fluorescence of the reagent blank a t GO "C increased exponentially after about 20 minutes, whereas a t 40 "C, the blank was fairly constant (even up to 120 minutes). Based on the greater stability of the O P T reagent, the increased specificity to 5-HTP, and the lower blank value, a period of 60 minutes a t 40 "C was selected as the optimum heating conditions. Effects of Reducing Agents. Various reducing agents have been used to prevent the oxidation of 5-hydroxyindole compounds during procedures used for their isolation and assay. Compounds such as ascorbate, cysteine, or glutathione apparently can react with O P T to form fluorescent products and the extent of this reaction is greater with higher temperatures of incubation (Table I). The effects of these compounds on the fluorescence of 5-HTP, 5-HT, or 5-HIAA reacted with O P T indicated that only in the case of 5-HIAA was there a net increase in fluorescence in the presence of added cysteine or glutathione, whereas in the case of 5-HTP, all three compounds significantly decreased the fluorescence (Table 11). Application of Method to Analyses of Tissue Samples and Various Standard Compounds. The data in Fig-
60
TIME (mm)
Figure 3. Time course of fluorophor formation at 40 OC The respective 5-hydroxyindoles (200 prnol) were incubated and analyzed as described under Experimental. The values reported have been corrected for blank readings
I
I
40
20
TIME (min)
Figure 4. Time course of fluorophor formation at 60 "C (See legend for Figure 3)
r'
5
0.50-
iii n
Ir
w
0.25-
C
0
0
I
I
I
I
I
20
40
60
80
100
L-5-HYDROXYTRYPTOPHAN (pmoles)
Figure 5. Fluorometric response of standard L-5-HTP in 0.01N HCI (0-0) and of L - ~ - H T Padded to a 5-HTP fraction isolated from tissue samples of rat brain minus proper blank (6-0). Standard conditions of assay were used (see Experimental)
ure 5 show the results obtained when varying amounts of L-5-HTP are added to a 5 - H T P fraction isolated from tissue by a method described elsewhere (15).After correcting for "blank readings," no difference was observed between these values and those for comparable amounts of standard 5-HTP in 0.01N HCl. Furthermore, the amount of fluorescence observed was linearly related to the amount of 5H T P in the sample. An indication of the degree of specificity of the assay procedure for 5-HTP is given in Table 111. The data show 5-HTP to be 60% more reactive than 5-HT or 5-HIAA under these assay conditions. I t should also be noted that 5-methoxytryptophan was about twice as reactive as 5-HTP.
DISCUSSION Due to the low content of 5-HTP in brain tissue, it has been necessary to develop a method with sensitivity in the picomole range. The reaction described by Maickel and Miller ( 5 ) between O P T and indole derivatives offered ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 1 , JANUARY 1975
9
Table I. Effect of Temperature on Reactivity of Ascorbic Acid, Cysteine, and Glutathione with O P T Relative fluorescence at 480 nma Reducing agent
Ascorbic acid L - Cysteine Glutathione
40
0.0070 0.0080 0.0099
so 'C 0.0214 i 0.0029 0.0579 * 0.0155 0.0457 i 0.0048
"c i 0.0002
* 0.0018
i 0.0008
82 =c 0.032 i 0.005 0.355 i 0 . 0 5 4 0.612 It 0.115
a Values given are means f std dev. of 3 determinations. The analysis a t 40 and 50 "C were carried out using the following conditions of assay: amount of O P T was 0.06 mg, length of time samples incubated a t specified temperature was 50 minutes, and final normality of HC1 was 8N. The amounts of reducing agent used in these two studies were: 0.43 pmol of ascorbic acid; 0.74 Fmol of cysteine, and 1.6 pmol of glutathione. The amount of reducing agent used in the studies a t 82 "C was: 0.21 pmol of ascorbic acid, 0.37 pmol of I-cysteine and 0.81 pmol of glutathione. The incubation time was 20 minutes and 0.1 mg of O P T was used. -
Table 11. Effect of Ascorbic Acid, Cysteine, a n d Glutathione Addition on O P T Assay of S-HTP, 5-HT, and 5-HIAA Relative fluorescence at 480 nma ~~~~~~
Reducing agent added
None (control) Ascorbic acid (0.21 pmol) L-Cysteine (0.37 pmol) Glutathione (0.81 p mol)
a
~~~
~~
5-HT
5-HTP
0.513 0.405 0.368 0.390
i 0.016
0.407 z 0.044 0.334 i 0. 005d 0.379 * 0.047 0.480 i 0.048
i 0. 005b
* 0.018*
5 -HlAA
0.284 0.218 0.405 0.547
i 0.042 i 0.008
i 0.058'' i 0.038' The values given are net relative fluorescence (i.e., sample fluorescence minus reagent blank fluorescence) and represent means f S.D. i 0,035'
of 3 determinations. The OPT-reaction was carried out at 82 " C for a total incubation time of 20 minutes. The samples, final volume of 150 pl, were prepared by combining 100 p1 of 12 hr HC1 containing 0.1 mg of O P T with 25 p l of the respective 5-hydroxyindole compound (100 pmoles) and 25 p1 containing the amounts of reducing agent given in the table above. * p-value < 0.001 with regards to control. c p-value < 0.01 with regards to control. d p-value < 0.005 with regards to control. All p-values are based on calculations using two-tailed Student " t " tests.
Table 111.Reactivity of Various Compounds with O P T Based on the Fluorescence at 480 nma Compound
Fluorescent yield relative t o 5-HTP
5-HTP 5-MTPb 5-HT 5 -HIAA Tryptophan 5- nilet hy 1tryptophan Kynurenine Anthranilic acid Tyrosine Nicotinic acid Nicotinamide Glycine
1.0 2.2 0.41 0.38 2 . 5 x 10-3 8.3 x 6 . 4 x lo-' 5 . 2 x 10-' No reactionc No reactionc No reaction' 2.2 x 10-7
Unless otherwise stated, the standard conditions of assay were used (see Experimental). All ratios are based on the fluorescence from equimolar amounts. * Heating time a t 40 "C was 50 minutes. c " o observed reaction when amount of compound assayed was 10 nmol.
what appeared to be the most sensitive method. The reaction conditions reported in their studies as well as in many other reports on the use of the OPT compound were designed for the assay of 5-hydroxytryptamine or 5-hydroxyindoleacetic acid. It, therefore, was necessary to determine the conditions for reaction of 5-HTP with OPT to give maximal fluorescent yields relative to the blank. The optimal conditions established are for a sample with a final volume of 150 ~1 contained in a cuvette made from glass tubing with an inner diameter of 3 mm. A value of 0.06 mg of OPT in the final volume used to read the fluorescence was found to be necessary if OPT was not to be a limiting component in the assay. This amount of OPT (equivalent to 0.4 mg/ml) is much greater than that reported by most other investigators. The only other study known to the authors where similar experiments were conducted with OPT is that by Komesu and Thompson (9). 10
They selected 0.5 mg/ml as being the optimal concentration of OPT in assaying for 5-HT. Unfortunately, a t this higher level of OPT and a t high reaction temperatures, the blank becomes extremely high, increasing with longer reaction times. Komesu and Thompson (9) overcame this problem through use of a chloroform wash. We found the blank could be stabilized by decreasing the reaction temperature to 40 O C without significantly affecting the fluorescence from 5-HTP. Even after 2 hours a t 40 "C, the blank did not increase significantly. Apparently a t the higher temperature and OPT concentration, the OPT itself can form a fluorescent derivative with a maximum wavelength (uncorrected) a t 440 nm. At higher temperatures, the fluorescence of the OPT reagent blank tends to increase exponentially after a period of time. The use of a lower reaction temperature (40 "C) made it necessary to go to a longer incubation time (60 minutes) in order for the reaction to go to completion. The optimal final concentration of HC1 was found to be 8N (Figure 2). These results are different than that reported for 5-HT by Thompson, Spezia, and Angulo (6). They reported a maximum a t about 6.5N, above which the fluorescent yield rapidly decreased. The difference in our results is attributed to their using the lower O P T concentration and reaction conditions reported by Maickel and Miller ( 5 ) in their studies. We have observed similar instability of the fluorophor when using low concentrations of OPT, high temperature, and heating periods greater than about 15-20 minutes. The instability of the fluorophor was even greater when sulfuric acid was used in place of HCl. The conditions selected as optimal in the assay of 5-HTP were: 0.06 mg of OPT in a cuvette volume of 150 p1; final normality of HCl of 8.0; reaction temperature of 40 "C; and incubation time of 60 minutes. After separating 5-HTP from other indoles, we have used these conditions with success in measuring endogenous levels of 5-HTP in samples from CNS tissue. A number of studies have recently been reported where various reducing agents have been added to samples of 5 -
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1 . JANUARY 1975
hydroxyindoles to stabilize them against auto-oxidation ( I O , 12, 13). The fact that ascorbic acid. cysteine, and glutathione can actually react with OPT t o form fluorescent products (Table I) and can have either an enhancement or diminishing effect on the fluorescence of a given 5-hydroxyindole compound (Table 11) indicate that caution should be exercised in the use of these compounds in assays for 5hydroxyindoles. The diminishing effect on fluorescence observed in the case of ascorbic acid is in agreement with the findings of Thompson e t al. (26) and the enhancement on fluorescence of 5-HIAA by cysteine is in confirmation with the findings by Korf and Valkenburgh-Sikkema 120). We have found that working in a dimly lit room, and keeping the samples in the dark as much as possible, is sufficient to prevent significant losses of 5-HT or 5-HTP from auto-oxidation and eliminates the need for the use of any antioxidants in the analyses of these two compounds.
LITERATURE CITED (1) W. Zimmerman, 2.Physiol. Chem., 189,4 (1930). (2)G. Klein and H. Linser, 2.Physiol. Chem., 205, 251 (1932). (3)A. R . Patton. J. Biol. Chem., 108,267 (1935). (4)G. Curzon and J. Giltrow, Nature (London), 173,314 (1954). (5) R . P. Maickel and F. P. Miller, Anal. Chern., 38, 1937 (1966). (6)J. H. Thompson, C. A. Spezia, and M. Angulo, Experientia, 25, 927
(1969). (7)J. H. Thompson, C. A. Spezia. and M. Angulo, lr. J. Med. Sci., 3 , 197 (1970). (8)J. H. Thompson, C. A. Spezia, and M. Angulo, Experientia, 26, 327 (1970). (9)N. N. Komesu and J. H. Thompson, Eur. J. Pharmacol., 16,248 (1971). (IO) J. Korf and T. Valkenburgh-Sikkema, Clin. Chim. Acta, 26, 301 (1969). (1 1) C. Atack and M. Lindqvist, Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol., 279,267 (1973). (12)C. A. Marsden, Comp. Gen. Pharmacol., 3, l(1972). (13)G. Curson and A. R. Green, Brit. J. Pharmacol., 39,653 (1970). (14)R. H. Cox, Jr., and J. L. Perhach, Jr., J. Neurochem., 20, 1777 (1973). (15)K. H. Tachiki and M. H. Aprison, manuscript submitted for publication. (16)J. H. Thompson, C. A . Spezia, and M. Anguio, Anal. Biochern., 31, 321 (1969).
RECEIVEDfor review May 2, 1974. Accepted September 9,
ACKNOWLEDGMENT
1974. This investigation was supported by Research Grant MH-03225-15 from the National Institute of Mental Health, U S . Public Health Service.
The authors gratefully acknowledge the excellent technical assistance of Roger Jackson.
Determination of Mercaptobenzothiazole (MBT) in Flotation Liquors by Solvent Extraction and Ultraviolet Spectrometry Michael H. Jones and James T. Woodcock CSlRO Division of Mineral Chemistry, P.O. Box 124, Port Melbourne, Victoria, Australia 3207
2-Mercaptobenzothiazole (MBT) is a constituent of some flotation collectors and there is a need to determine residual MBT in plant liquors and effluents. MBT can be determined by UV spectrometry at 329 nm in a chloroform extract obtained after acidifying the liquor to less than pH 2 or after adjusting the pH to 7-8 with ammonium acetate. MBT in chloroform has a molar absorptivity of 28100 f 200 liter mole-‘ cm-‘ and obeys Beer’s law up to 9 mg/l. At pH 1, with an aque0us:organic ratio of 1:1, 99% of the MBT is extracted in one stage, and the absorbance of the chloroform extract is directly proportional to MBT concentration in the original aqueous solution (MBT concentration (mgll.) = 5.95 X absorbance in 1-cm cells). The nominal detection limit is 0.1 mg/l. but this can be extended to 0.01 mg/l. by using higher aqueous:organic ratios. Few compounds interfere, although cuprocyanide interfered with extraction from acid solution but not from ammonium acetate solution. Ore slimes in the aqueous phase did not interfere with extraction.
A knowledge of residual reagent concentrations in a flotation pulp may give a better understanding of the process and may be a guide to reagent addition rates. Anti-pollution measures may also require a knowledge of residual reagent concentrations in flotation plant effluents. 2-Mercaptobenzothiazole (MBT) or 2(3H)-benzothiazolethione is widely used as a flotation collector. I t is marketed under such names as MBT, Captax, and Flotagen, and is a major constituent of reagents in the 400 series of the American Cyanamid Company.
In a previous paper ( I ) it was shown that direct UV spectrometry of an aqueous filtrate from a flotation pulp could be used to measure MBT concentrations up to 12 mg/l. using a 1-cm cell. Cuprocyanide interfered when more than 20 mg/l. was present. Suspended matter in the liquor also interfered. Because MBT is more soluble in organic solvents than in water, it was thought that it should be possible to extract M B T from aqueous solution and measure the absorbance of the extract. Koch (2) studied the UV absorbance of rubber accelerators in chloroform and benzene, and Kress ( 3 )applied this information to the analysis of accelerator-rubber mixtures by measuring the UV absorbance in chloroform. In this work, the main effort was directed to the use of chloroform which seemed to be the most suitable solvent. EXPERIMENTAL Equipment. Spectral scans were obtained with a Unicam SP800A recording spectrophotometer using stoppered 1-cm quartz cells and linear wavelength and absorbance presentation. Instrument calibration was checked using holmium oxide glass and aqueous solutions of potassium dichromate and nitrate ( 4 ) . Mercaptobenzothiazole. For much of the work, pure 2-mercaptobenzothiazole prepared for the previous study ( I ) was used. This contained C, 50.02; H, 2.99; N, 8.22; S, 38.5; melting point 180 “C (calculated for C7HsNS2: C, 50.27: H, 3.01: N, 8.38; S, 38.34). A second batch, prepared 12 months later, analyzed C, 50.25; H, 3.05; N, 8.09; S, 38.5. Other Reagents. AR chloroform was used without purification. Flotation chemicals were used “as received.” Reagents that were not completely soluble in water were made up a t 500 mg/l., filtered. and the filtrate was used.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, JANUARY 1975
11
