(9) R. Long, "Studies on Polycyclic Arom. Hydrocarbons in Flames", €PA R372 020, June 1972. (10) L. Wallca, Environ. Sci. Techno/., 3, 948 (1969). (11) N. P. Buu-Hoi, P. Jacquignon, J. P. Hoeffinger, and C. Jutz, Bull. SOC. Chem. Fr., 2514 (1972). (12) L. J. Bellamy, "The Infra-red Spectra of Complex Molecules", 2nd ed., John Wiley & Sons, New York, 1964, pp 127-129. (13) J. Michl, R . Zahradnik, and P. Hochmann. J. fhys. Chem., 70, 1732 (1966). (14) R. Zahradnik, M. Tichy, and D. H. Reid, Tetrahedron, 24, 3001 (1968). (15) D. H. Reid and W. J. Bonthrone, J. Chem. SOC.5920 (1965).
(16) W. C. Heuper, P. Kotin, E. C. Tabor, W. W. Payne, H. Falk, and E. Sawicki, Arch. fatho/., 74, 89 (1962). (17) L. L. Ciaccio, R . L. Rubino, and J. Flores, Environ. Sci. Techno/., 8, 935 (1974).
RECEIVEDfor review December 2,1974. Accepted March 3, 1975. This work was funded by the B. F. Goodrich Company-United Rubber, Cork, Linoleum and Plastic Workers Joint Occupational Health Program.
Determination of Anionic Surfactants with Azure A and Quaternary Ammonium Salt Lawrence K. Wang and Pedro J. Panzardi Department of Chemicaland EnvironmentalEngineering,Rensselaer Polytechnic Institute, Troy, N Y
The common surfactant portion of synthetic detergents is an anionic surfactant named linear alkylate sulfonate (LAS), shown in Figure 1. Analysis of the residual concentration of LAS in water and wastewater is of importance to environmental chemists and engineers. The Methylene Blue method ( I , 2) and the carbon adsorption method ( I ) are the recommended standard methods for the analysis of LAS or other anionic nonsoap surface-active agents. Both the standard methods involve the use of spectrophotometer, and are extemely time consuming. Recently, Wang ( 3 ) has proposed a modified Methylene Blue method for the determination of LAS as well as other anionic nonsoap surfactants with a spectrophotometer. Wang simplified the number of solvent extractions from seven to two (including the extraction for a blank) and reduced the possible interference caused by chloroform-extractable pollutants. Sufficient experimental data have been obtained ( 4 ) to conclude that the modified Methylene Blue method is more precise than the standard Methylene Blue method. For the environmental water quality control, there is a need to establish a standard titration method for rapid analysis of anionic surfactants in the field. The objective of this paper is to introduce the two-phase titration techniques which can be used for determining LAS and other anionic nonsoap surfactants. The titration techniques would be convenient to be used in the field due to the fact that there is no need for sophisticated instrumentation (not even a colorimeter or simple photometer). In the next section, a general survey of the two-phase titration is presented. In the Experimental section, the authors offer a new two-phase titration involving the use of Azure A and Methyl Orange as primary dye and secondary dye, respectively. Its advantages and limitations are also discussed.
BACKGROUND Two-phase titration using Methylene Blue was initially proposed by Weatherburn in 1950 (5). In his method, the sample containing anionic nonsoap surfactants is first treated with the Methylene Blue reagent and chloroform. After extraction, a blue-colored, chloroform-soluble dyesurfactant complex is formed in the chloroform phase. The treated sample is then titrated with a solution of alkyltrimethylammonium chloride with intermittent vigorous shaking until the end point is reached (i.e., the blue color is completely discharged from the chloroform layer). Weath1472
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
erburn's method has been tried by the authors. It is difficult to detect the end point because the blue color in the top aqueous layer will reflect to the bottom chloroform layer. In 1954, Edwards (6) offered a modified two-phase titration method for use on sewage, to eliminate the interference by proteins, hardness, and soap. Azophloxine dye was substituted for Methylene Blue, hexane for chloroform, and cetyltrimethylammonium bromide for alkyltrimethylammonium chloride. The titration end point is reached when color appears in the hexane layer. A drawback of Edward's method is the necessity of using a centrifuge to facilitate the phase separation. Turney suggested an alkaline Methylene Blue two-phase titration method in 1965 (7). He used Methylene Blue reagent as complexation agent, chloroform as organic solvent, and Hyamine 1622 (diisobutylphenoxyethoxyldimethylbenzylammonium chloride) solution as titrant. The sample containing anionic surfactant is at first treated with the Methylene Blue reagent, chloroform, and 15% sodium hydroxide, then titrated with Hyamine 1622, shaking after each addition. The organic solvent layer is initially blue in color, and changes gradually to red-violet to pink. The aqueous layer, however, becomes colorless at the end point. Hyamine 1622 was later adapted as cationic titrating solution by Reid (8, 9) for his new two-phase titration method using the mixed indicators (dimidium bromide and Disulphine Blue VN). Reid also selected chloroform as organic solvent. The anionic surfactant sample containing all necessary reagents is titrated with Hyamine 1622 solution until the pink is discharged from the organic solvent layer. The end point is reached before the color in the organic solvent layer turns to distinct blue. Wang et al. (10) recently developed an indirect twophase titration method for the identification of LAS in water. Their indirect method uses cetyldimethylbenzyl ammonium chloride (DCBAC) in excess amount to form a complex with the water-soluble anionic surfactant, and uses Methyl Orange (MO) as an indicator (reacts with excess CDBAC) in the presence of chloroform. The color of the CDBAC-MO complex in the chloroform phase is yellow. This water-chloroform two-phase mixture is then titrated with sodium tetraphenylboron (STPB) reagent with intermittent shaking to ensure equilibrium between the chloroform and the aqueous phases. The disappearance of the yellow color in the bottom chloroform layer indicates
"=I=
0 Na
ALKYLATE SULFONATE
P
Ida
0
0
Figure 2. Blue colored complex of anionic surfactant with Azure A
Figure 1. Simplified molecular structure of linear alkylate sulfonate
&AS)
Table I. Evaluation of Direct Two-Phase Titration Using Carbon Tetrachloride as Organic Solvent the end point of the titration. The actual concentration of anionic surfactant, such as LAS, is given by the difference between the amount of the CDBAC added to the sample and the amount of S T P B needed for titration. This procedure is reliable for LAS analysis in both fresh and sea water samples ( 10). Subsequent sections introduce the authors' two-phase titration method which is essentially a modification of the previously published methods (5-10). Azure A has been suggested by Steveninck and Riemersma (11) and Wang (12) to replace Methylene Blue in colorimetric determination of anionic surfactants. Research conducted by Wang (12) confirms that the rate of phase separation is a t least five times faster when Azure A is substituted for Methylene Blue in the colorimetric method. Accordingly, Azure A was selected as primary dye in the authors' two-phase titration method. Methyl Orange was selected as secondary dye to measure any excess amount of cationic titrant; thus the end point of titration can be accurately detected. The authors' method also has the flexibilities of selecting either chloroform or carbon tetrachloride as organic solvent, and selecting one of suggested pure quaternary ammonium compounds as cationic titrant. Linear alkylate sulfonate, an anionic surface-active agent, is suggested to be used for preparing calibrating solution for environmental water quality control.
EXPERIMENTAL Principle. The water sample to be analyzed is introduced in a separatory funnel where Azure A (AA), buffer, and organic solvent (chloroform or carbon tetrachloride) are added to it. With extraction, the organic solvent layer acquires a color because of the formation of a AA-LAS complex as shown in Figure 2. (Note: The color of organic solvent layer will be blue for chloroform and will be violet for carbon tetrachloride.) Such AA-LAS complex becomes soluble in the organic solvent upon adjusting the pH to 3. This two-phase mixture is then treated with a cetyldimethylbenzyl ammonium chloride (CDBAC) reagent with intermittent shaking to ensure equilibrium between the organic solvent and the aqueous phases. As CDBAC reacts and displaces the AA molecules, the bottom organic solvent phase starts to turn colorless. This is because the AA itself is not soluble in organic solvent. Near the end point of the titration, the top aqueous layer is blue in color, while the organic solvent layer turns colorless. Some reflection of the color to the bottom organic solvent layer may occur and this makes the end point difficult to determine. A modification to the test method can be made by adding Methyl Orange to the sample near the end point of the titration where the organic solvent layer is nearly colorless. With this modification, the organic solvent phase acquires a greenish-yellow color (for chloroform) or a yellow color (for carbon tetrachloride) when the end point is reached. This is due to the fact that Methyl Orange forms an organic solvent-soluble complex with CDBAC but not with AA. A background sample containing water, buffer, and AA but no surfactant is recommended to compare the color a t the end point.
Aqueous samples SO u.4 U S 2 2 0 p g LAS 5 2 0 p g LAS
Volume of 50 mg/l. CDBAC solution s p e n t i n t i t r a t i o n , ml
Average
2.20 2.30 2.25 2.30 2.25 2.25 2.30 2.30 2.20 2.30 2.26
5.85 5.80 5.65 5.75 5.80 5.75 5.70 5.70 5.80 5.65 5.74
14.35 14.30 14.30 14.30 14.35 14.30 14.30 14.25 14.30 14.40 14.32
Relative standard deviation, o/c R e l a t i v e e r r o r , ($
7.23 3.78
4.56 4.19
2.77 1.27
Apparatus. The titration apparatus and some glassware including separatory funnels (125 ml, preferably with inert Teflon stopcocks) are required. Reagents. Required reagents for routine analysis are: 50 mgh. of cetyldimethylbenzyl-ammonium chloride, 0.10% of Methyl Orange solution, 0.10% azure A solution, reagent grade of chloroform (or carbon tetrachloride), 0.5M of citric acid, and 0.2M of disodium hydrogen orthophosphate solution. Buffer solution can be prepared monthly by mixing 250 ml of 0.5M citric acid and 250 ml of 0.2M disodium hydrogen orthophosphate solution together. A 50-mgL. linear alkylate sulfonate solution is required for the preparation of a calibration curve for environmental water quality control. Once the calibration is established, the linear alkylate sulfonate solution is not needed. Procedure. a ) Preparation of Calibration Curve. Prepare a series of separatory funnels with 0, 2, 4,6, 8, 10, 15, 20, 25, and 30 ml of the 50-mgh. LAS solution. Add distilled water to make total volume 30 ml in each separatory funnel. Treat each sample as described below in procedures d ) through f). Plot a straight-line calibration curve consisting of wg LAS vs. ml of 50-mgh. CDBAC solution spent in titration. [Note: a calibration curve can be done using anionic surfactants other than LAS.] b ) Volume of Sample. Select the volume of the water to be treated on the expected LAS concentration such that each sample containing less than 1500 Kg LAS (or equivalent) is placed in a separatory funnel for test. c ) Pipette an aliquot amount of the anionic surfactant sample into a separatory funnel, dilute to 30 ml with distilled water. d ) Add 5 ml of buffer solution, 20 drops of Azure A 0.1% solution, and 30 ml of organic solvent (chloroform or carbon tetrachloride). e ) Titrate this solution in the separatory funnel with 50 mgh. of CDBAC soltuion by adding small amounts, restoppering, and shaking. f) Continue titration until the color (blue for chloroform, or violet for carbon tetrachloride) in the organic solvent layer is imperANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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Table 11. Evaluation of Direct Two-Phase Titration Using Chloroform as Organic Solvent Aqueous samples
SO
u g LAS
200 ug I A S
300 u g LAS
500
wu s
Average
2.10 2.13 2.12 2.10 2.10 2.15 2.10 2.13 2.12 2.15 2.12
5.30 5.27 5.32 5.30 5.30 5.30 5.30 5.30 5.28 5.30 5.30
7.97 7.95 7.95 7.90 7.95 7.95 7.95 7.95 7.90 7.95 7.94
12.95 12.95 12.90 12 -95 12.90 12.95 12.90 12.98 12.95 12.95 12.94
Relative standard deviation, % ’ Relative e r r o r , %
1.60 1.50
3.55 1.55
2.37 1.42
1.84 0.83
Volume of 50 mg/l. CDBAC solution spent in titration, m1
ceptible. Add 10 drops of Methyl Orange 0.1% solution and continue titrating until the bottom organic solvent layer changes color (to greenish yellow for chloroform, or to faint yellow for carbon tetrachloride). Record the amount of the 50 mg/l. CDBAC solution required for titration. g) For unknown samples, a measure of LAS present in the sample is determined from the calibration curve described in procedure ( a ) ,and the value is given in Fg LAS per sample.
Calculation
mg/l. as LAS =
pg as LAS
ml of sample
(Anionic surfactants other than LAS can also be used as a basis for determining the amount of surfactants in the water sample.)
RESULTS AND DISCUSSION Evaluation data were obtained by titrating the distilled waters containing known amounts of linear alkylate sulfonate (Le., LAS) and Azure A (i.e., AA) with 50 mgh. cetyldimethylbenzylammonium chloride (Le., CDBAC) solution. LAS was supplied in liquid form by the Analytical Quality Control Laboratory, Envircrmental Protection Agency, 1014 Broadway, Cincinnati, OH; AA was supplied in powdered form by Fisher Scientific Company (Catalog No. A970), Rochester, NY; and CDBAC (Trade name: cetol) was manufactured by Fine Organics Inc., 205 Main Street, Lodi, NJ. In order to evaluate the direct two-phase titration method, three different concentrations were used for extensive investigation. Ten independent determinations were performed for each LAS concentration selected. The results are summarized in Tables I and 11. From the average titration results listed in Tables I and 11, two straight-line calibration curves (one for chloroform and one for carbon tetrachloride) can be obtained with the following data points: 50 m o l l . CDBAC titrant, m l
LAS in sample, w g
0 80 200 220 300 500 520
CCk
C1iClj
0 2.26
0 2.12 5.30
...
5.74
...
...
14.32
...
7.94 12.94
...
Using the experimental data presented in Table I, the precision and accuracy of the test method involving the use of carbon tetrachloride as the organic solvent were also determined. For example, a sample containing 2.67 mg/l. of LAS (i.e., 80 pg LAW30 ml) in distilled water was determined with a relative standard deviation of 7.23% and a rel1474 * ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
ative error of 3.78% for ten independent determinations. A sample containing 16.67 mgh. of LAS (Le., 520 pg LAS/SO ml) was determined, with a relative standard deviation of 2.77% and a relative error of 1.3% for ten independent determinations. Table I1 indicates the experimental data of two-phase titration using chloroform as the organic solvent. The highest relative standard deviation and relative error were determined to be 3.55% and 1.55%’ respectively. Chloroform is a better solvent (or extractant) compared to carbon tetrachloride. Nevertheless, either chloroform or carbon tetrachloride is a good organic solvent for the proposed twophase titration technique. An environmental chemist or engineer can use either of them depending upon the availability. The precision and accuracy of the standard methylene blue method have been evaluated by 110 laboratories ( I ) . It has been reported that a) a synthetic unknown sample containing 270 mgh. LAS in distilled water was determined with a relative standard deviation of 14.8% and a relative error of 10.6%; b) a tap water unknown sample to which was added 480 mg/l. LAS was determined, with a relative standard deviation of 9.9% and relative error of 1.3%; and c) a river water unknown sample to which was added 2.94 mg/l. was determined, with a relative standard deviation of 9.19% and a relative error of 1.4%. The results obtained with the authors’ method are good but comparison with the standard Methylene Blue method would be valid only for the LAS concentration common to both (2.67 mg/l. or 80 pg/30 ml of LAS analyzed by the authors’ method compared to 2.94 mg/l. LAS analyzed by the standard Methylene Blue method). From the evaluation data, it can be concluded that the authors’ direct two-phase titration method would be either slightly more precise or equally as precise and accurate compared to the standard Methylene Blue method. The former, however, is much simpler and less time consuming than the latter. There is also an option in selecting the cationic titrating solution. In fact, any pure quaternary ammonium salt can be used. The authors, however, recommend either cetyldimethylbenzylammonium chloride, or cetyltrimethylammonium bromide be chosen for the titrant preparation. Both quaternary ammonium salts are white, free-flowing powders, cationic in nature, and 100% pure. They can be ordered directly from Fine Organics Inc., Lodi, NJ. One limitation of the two-phase titration method using Azure A presented here is one common to all the mentioned methods (1-12). None of the methods is capable of resolving and identifying the different types of anionic sur-
factants being analyzed. The surfactant analysis can only be expressed as mgA. as Azure A Active Substances (AAAS).
(IO) L. K. Wang. J. Y. Yang, and M. H. Wang, J. Am. Water Works Assoc., 67(1), 6-8 (1975). (11)J. V . Steveninck and J. C. Riemersma, Anal. Chem.. 38, 1250-1251 (1966). (12)L. K. Wang, "Improved Colorimetric Methods and Field Test Kits for Analyzing Anionic Surfactants in Water and Wastewater," Tec. Rep. No. ND-5296-M-4, Calspan Corporation, Buffalo, NY, pp 1-5 1, August
LITERATURE CITED (1)"Standard Methods for the Examination of Water and Wastewater", 13th ed., American Public Health Association, Washington, DC, 1971,p 339. (2)"Methods for Chemical Analysis of Water and Wastes", U.S. Environmental Protection Agency, Washington, DC, 1974,p 157. (3)L. K . Wang, J. Am. Water Works Assoc., 67 (1).19-21 (1975). (4)L. K . Wang, W. W. Shuster. and P. J. Panzardi, J. Am. Water Works Assoc., 67 (4),182-184 (1975). (5) A. S.Weatherburn, J. Am. OilChem. Soc., 28, 233-235 (1950). (6) G. R . Edwards and M. E. Ginn. Sewage Ind. Wastes, 26, 945-953 (1954). (7) M. E. Turney and D. W. Cannell, J. Am. Oil Chem. Soc., 42, 544-546 (1965). ( 8 ) V. W. Reid, G. F. Longman. and E. Heinerth. Tenside, 4(9). 292-304 (1967). (9) V. W. Reid, G. F. Longman. and E. Heinerth, Tenside, 5(3-4), 90-96 (1968).
1973.
RECEIVEDfor review November 18, 1974. Accepted March 12,1975. This research was supported by the Economic Development Administration of Puerto Rico, and an unrestricted research grant (RPI Project No. IBN 25) from Gruman Aerospace Corporation, Long Island, NY, through the School of Engineering, Rensselaer Polytechnic Institute, Troy, NY. The authors thank Arthur A. Burr, Rensselaer Professor, School of Engineering, RPI, for making this research possible.
Wet Ashing of Some Biological Samples in a Microwave Oven Adel Abu-Samra, J. Steven Morris, and S. R. Koirtyohann Environmental Trace Substances Research Center, University of Missouri, Columbia, MO 6520 1
Problems associated with methods for the destruction of organic matter prior to metals analyses have been the subject of many papers and are probably covered most thoroughly by Gorsuch ( I , 2). Wet ashing methods seem to have gained greatest acceptance among workers interested in trace metals analyses. Disadvantages of wet ashing include the close and constant operator attention which is required, the need for special hoods to handle perchloric acid fumes safely, and the danger of explosion if established procedures are not strictly followed. In certain cases, i.e., neutron activation analysis using short lived isotopes, the elapsed time can also be a limitation. These problems can be minimized if wet ashing is done in an adapted inexpensive commercial microwave oven. Acid mixtures are heated internally by the oscillating electromagnetic field, resulting in very rapid, safe, and efficient ashing. Although microwave heating has been reported as an efficient sample drier ( 3 ) ,this appears to be the first account of a modified microwave system suitable for wet ashing.
Procedure. The exact procedure depends to some extent on sample size which has ranged from a few mg to 1 gram in our experiments. The container selected, the volume of acids added, and the time required for ashing vary but none seem to be critical. Typically, 0.5-gram samples of dried plant or animal tissue are placed in 125-ml Erlenmeyer flasks and 10 ml of the nitric-perchloric acid mixture added to each. The flasks are then placed inside the oven and ashed for a preselected time. Progress of the digestion can be followed visually and it is usually terminated when perchloric acid fumes appear (approximately 200 OC). The manufacturer suggests that the oven should not be operated empty or nearly empty for extended periods because of the risk of damage to the magnetron. Therefore, a beaker containing 100 ml of water is placed inside the cavity whenever the total acid volume is small. Procedures for evacuation and trapping of acid fumes required considerable attention. Evacuation by water aspirator, a small vacuum cleaner, and a 6-inch diameter drum fan (Dayton Model 4C006), all were employed. A trap to remove acid fumes from the exhaust gases prior to venting is normally used. Trap materials have included solid CaO, solid CaC03, liquid NaOH, and a water spray.
EXPERIMENTAL Apparatus. Microwave oven: The oven was purchased in a local department store (Montgomery Ward Signature Microwave Oven) and is similar to many other brands sold for home use. It is rated at 600 watts and has a cavity about 24 X 36 X 39 cm. T o protect the inside of the oven from very corrosive acid fumes, a locally made Plexiglas box was placed inside the cavity. Large pieces of metal should not be placed in the oven, but the small hinges needed for the door and the screws to fasten them have caused no problem, probably because they are located in the corner of the cavity. A 1-inch diameter hole was drilled through the side of the oven and the liner to accommodate a glass exhaust port which is connected to a suitable evacuation system. The modified unit was checked using a Narda microline Model 8200 electromagnetic leakage monitor and found to be free of microwave radiation leakage. Reagents. A 4:l mixture of reagent grade nitric and perchloric acids was used for most ashings. In a few cases, nitric acid and 30% hydrogen peroxide were used.
Table I. Analysis Results of 1577 Bovine Liver a n d 1571 Orchard Leaves by Flame Atomic Absorption after Microwave Oven Wet Ashing 1577 Bovine Liver, * g / g
1 Zn
Cu
132 193
2 131 194
3 131 196
4
(av)
(NBS value)
131
(131) (194)
(130 10) (193 i 10)
193
1571 Orchard Leaves, w g l g .___~__
Pb
Zn Cu
1 42 27 11.3
2 45 32 11.8
3 45 21 11.7
4 46 22 11.8
(av)
(NBSvalue)
(44) (26) (11.6)
(45 i 3 ) (25 3) (12 i 1)
ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
*
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