Electrophoretic Separation of Alkytsulfate, Alkylbenzenesulfonate, and Alkylethoxysulfate Homologs, Using Aqueous Dioxane Agarose Gels John R. Bodenmiller and Howard W. Latz Department of Chemistry, Ohio University, Athens, Ohio 45701
Electrophoresis is used for the separation of members of homologous series which are common to anionic surfactants. The separation of members of homologous series could not be obtained in aqueous agarose gels. This is due to the monomer-micelle equilibrium which exists in these solutions. The effects of the surrounding media on the monomer/micelle ratio are demonstrated. Separations of homolo ous members of synthetic and commercial mixtures ofalkylbenrenesulfonates and alkylsulfates, as well as commercial alkylethoxysulfates and alkylarylethoxysulfates are obtained in media which are not conducive to the existence of micelles. These electrophoretic separations are carried out using aqueous agarose gels containing up to 50% dioxane. Migration distances of 25 to 30 cm and time periods of 2.00 to 2.75 hours are required to obtain complete resolution of adjacent members, with ionic weights not greater than 325, of these homologous series. Greater migration distances are needed to obtain complete resolution of heavier adjacent homologs. After separation, the components of the mixture are detected with pinacryptol yellow and are identified by comparing their migration distances with standards run at the same time. Separation profiles are obtained by direct photometric scanning of the fluorescent pinacryptol yellow-surfactant adducts. QUALITATIVE AND QUANTITATIVE analysis, of the hydrophobic portion of members of the homologous series which are common to anionic surfactants, is a problem which still lacks a satisfactory solution. Early work on the separation of homologs of anionic surfactants was done by Holness and Stone ( I ) , Franks (2), and Borecky (3), using paper chromatographic methods. Puschel and Prescher (4) published a more recent paper using paper chromatographic methods for the separation of alkylsulfates, alkylsulfonates, and various other homologous series of aliphatic sulfonates. The running times for such paper chromatographic methods are generally eight to sixteen hours. Another popular approach to this problem has been by gas chromatography (5-10). This method requires a chemical or pyrolytic conversion of the nonvolatile salts to more volatile compounds that can be separated in the gas chromatograph. The separation and identification of cationic surfactants by paper electrophoresis was attempted by Fumasoni, Mariani, and Torraca ( I I ) , (1) H. Holness and W. R. Stone, Nature, 176,604 (1955). (2) F. Franks, ibid., p 693. (3) J. Borecky, Collect. Czech. Chem. Commun., 28, 229 (1963). (4) F. Puschel and D. Prescher, J. Chromatogr., 32, 337 (1968). (5) J. J. Kirkland, ANAL.CHEM., 32, 1388 (1960). (6) T. H. Liddicoet and L. H. Smithson, J. Amer. Oil Chem. SOC., 42, 1097 (1964). (7) L. L. Lew, ibid., 44, 359 (1967). (8) C. B. Puchalsky, ANAL.CHEM., 42,803 (1970). (9) S. Siggia and L. R. Whitlock, ibid., p 1719. (10) P. Bore and F. Degremont, Rev. Fr. Corps Gras, 17, 315 (1970). (11) S. Fumasoni, E. Mariani, and G . Torraca, Chem. Znd. (London), 75,69 (1956). 1354
and by Noshiro, Izawa, and Kimura (12) but the migration rate varied with the sample concentration. Latz and coworkers investigated the electrophoretic properties of anionic surface active agents in aqueous agar and acrylamide gels (13). Their results indicated that the relative mobilities of surfactant types were dependent on pH and buffer type. This paper presents an electrophoretic method for the separation and identification of members of homologous series which are common to anionic surfactants. Explanations are offered for the electrophoretic behavior of surfactants under various conditions. The present authors are aware of no other successful attempts to apply electrophoresis to the separation and identification of the members of various homologous series which are common to anionic surfactants. Specifically, the method was applied to alkylbenzenesulfonates (ABS), alkylsulfates (AS), alkylethoxysulfates (AES), and alkylarylethoxysulfates (AAES), but it can be applied to similar anionic series and also to cationic surfactants. The electrophoresis was carried out using aqueous dioxane agarose gels. EXPERIMENTAL
A Desaga/Brinkmann Apparatus for ThinLayer Electrophoresis (Brinkmann Instruments Inc., Westbury, N. Y.)was used for preliminary studies. Other investigations, where indicated, were done using an electrophoresis cell of similar design except the length was such that a 1Cinch (35.6-cm) X 8-inch (20.3-cm) glass plate was used instead of the 20-cm X 20-cm plate used in the former. Power for electrophoresis was supplied by a Savant constant voltage source (Savant Instruments Inc., Hicksville, N. Y .). The wicks can be divided into three components, paper, gauze, and cellophane. A paper wick (Brinkmann Instruments Inc.) extends from the bottom of the upper buffer chamber to the lower buffer chamber. A paper wick also extends from the front bottom edge of the upper buffer chamber and overlaps the gel layer 1.5 cm. A paper carton wick (Brinkmann Instruments Inc.) was placed in the buffer chamber. The paper wicks and paper carton were then covered by a post-op sponge, SR-8254 (The Seamless Rubber Co., New Haven, Conn.). A second carton wick was placed in the upper buffer chamber on top of the gauze sponge. A strip of cellophane, obtained by splitting 1 l/s-inch dialyzing tubing (10886 Will Scientific Inc., New York, N. Y,) was placed on the under side of the wick so that contact between the wick and gel was made by the cellophane strip. The spectrophotofluorometer with thin layer scanner attachment was the same as that used by Madsen and Latz (14). Materials, The agarose, Catalog No. J-24043, was purchased from the Fisher Scientific Co., New York, N. Y. Apparatus.
(12) T. Noshiro, Y . Izawa, and W. Kimura, Kogyo Kaguku Zasshi, 68, 1587 (1965). (13) H. W. Latz and Coworkers, Union Carbide Corp., Tarrytown, N. Y., unpublished work, 1965. (14) B. C. Madsen and H. W. Latz, J. Chromatogr., 50, 288 (1970).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
and was manufactured by L'Industries Biologie Francaise S.A. The 1-ABS and p-(1-ethyldecy1)benzenesulfonate (p-3-12-S) were synthesized with H2S04,using standard methods from alkylbenzenes purchased from Eastman Organic chemicals, Rochester, N. Y. The 1-AS were prepared by the method of Dreger, Keim, Miles, Shedlovsky, and Ross ( I S ) from alcohols purchased from Eastman Organic Chemicals. The ABS mixtures, 215, 225, and 230, were synthesized with H2S04, using standard methods, from linear alkylbenzenes supplied by Monsanto Co., St. Louis, Mo. The numbers refer to the alkylate numbers assigned to the alkylbenzene mixtures by Monsanto Co. The homolog distribution of these alkylbenzenes are given in Table I. The sources of other commercial surfactants are given in Table 11. Procedure. PREPARATION OF THE GEL. The procedure for the preparation of a 50% dioxane gel is presented. All other preparations were similar with the appropriate substitutions. The gel was prepared by adding 1.08 grams of agarose to 45 ml of 0.020M, pH 6 phosphate butler in a 250-ml beaker. The beaker was covered with a watch glass and suspended in a water bath which was heated to boiling temperature. After the agarose was completely dissolved, the gel solution was removed from the water bath and 45 ml of dioxane was quickly added by pipet to the partially covered beaker. The contents of the beaker were mixed by a slight circular motion while the dioxane was added. The hot solution was then either heated or cooled to 60-70 "C and then poured on a 35cm X 20-cm X 2.5-mm glass plate. Prior to pouring the gel, the glass plate was placed on a level surface and other 6-mm thick glass plates were arranged so as to build a form for the gel. The seams were sealed with paraffin, which was accomplished by quickly moving a medicine dropper of hot paraffin down the seam. One to two minutes after the gel was poured, the glass forms were removed by carefully lifting up the side opposite the gel and then pulling it away from the gel. In this manner, the paraffin wax remained on the form and not on the gel plate. A strip of gel was then cut away from the two sides of the plate so that there was about 1.5 cm between the gel and the edge of the glass plate. A template containing eight holes, 2.0 cm apart was placed over the plate and a melting point capillary (1.5-mm diameter) was used to cut the gel. The gel cylinders were then removed by applying a vacuum with a second capillary connected to an aspirator. The holes were usually made about 4 cm from one end of the plate. This gives a gel which is 0.010M in pH 6 phosphate, 1.2% agarose, 50% dioxane and 50% water, and is about 1.3 mm thick. ELECTROPHORESIS OF THE SAMPLE. A light petroleum grease was applied to the aluminum cooling block in order to get better contact between the glass and aluminum surfaces. The prepared plate was placed on the cooling block and the wicks, which were already in the buffer vessels, were placed about 1.5 cm on the ends of the gel layer. The buffer vessels contained pH 6, 0.010M phosphate in pure aqueous solution. The circulation of the cooling water was then started. Sample, 1.6 p l , was deposited in the prepared holes by the use of a blunt needle microsyringe. The cover of the cell was closed and the appropriate potential was applied to the electrodes. The anode and cathode ends were altered after each run and the buffer was replaced after every fourth run. DETECTION.After completion of the run, the plate was removed from the cell and was placed on a flat surface. A saturated solution of pinacryptol yellow was applied slowly until it completely covered the second half of the plate. This was allowed to stand for 15 minutes and then removed (15) E. E. Dreger, G . I. Keim, G . D. Miles, L. Shedlovsky, and J. Ross, Itid. Eng. Chem., 36, 610 (1944).
Table 1. Homolog Distribution of Monsanto Phenylalkanes, Per Cent0 Alkyl chain ClO c 1 1
c 1 2
Ct3
215 7 56
33 4
225 4 42 38 15
1 237 242 Technical Data Sheet 1-269 Monsanto Company. c 1 4
...
Av mol wt a
230 , . .
4 24 49 23
259
Table 11. Names and Sources of Commercial Surfactants Sample Manufacturer Source Ucane- 11 Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y . Ucane- 12 Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y . Union Carbide Corp. Union Carbide Corp., Ucane- 13 Tarrytown, N. Y , Tergitol 15-s-4.6A Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y . Tergitol 45-s-3A Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y. Tergitol 1'5-s-3S Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y . Tergitol Anionic 4 Union Carbide Corp. Union Carbide Corp., Tarrytown, N. Y . Neodol 25-s-3A Shell Corporation Intermediary G. A. F. Alipal CO 436 Intermediary P.D. L.S.-l Alcolac Chem. Corp. Intermediary L.S.-2 Sigma Chemicals, St. Louis, Mo.
by tilting the plate. The pinacryptol yellow solution remaining on the surface was rinsed off by water from a wash bottle. A solution of 3 M NaCl was poured on the plate and allowed to stand for 5 minutes. The purpose of the chloride was to quench the background fluorescence of the pinacryptol yellow. The fluorescent sample spots were detected under ultraviolet light. If the separation profile was to be recorded, the following preparations were made. The gel was cut across the center midway between the two ends. The plate was submerged in water and the gel half containing the samples was floated onto a 20-cm X 20-cm X 1-mm glass plate. This transference was necessary because only 20-cm X 20-cm plates will fit into the thin-layer scanner. This was accomplished by carefully sliding the 20-cm plate between the gel and the 35-cm plate and presented no problems. After the 20-cm plate was carefully removed from the water tank, it was tilted at a slight angle against an absorbing substance to remove surface water. The gel was positioned with a straight edge, by sliding, so that the near edge was 2.54 cm from the end of the plate. A 1-cm strip was then trimmed off the other three sides and the plate was allowed to stand for 30 minutes. After this time was completed, the gel plate was submerged in water for approximately 30 minutes to remove excess pincryptol yellow from the gel. Free water was removed and the gel was repositioned in the same manner as before. RECORDING OF SEPARATION PROFILES. The yellow and orange-yellow fluorescence, of the pinacryptol yellowanionic surfactant adduct, made possible the recording of emission profiles for the separation. The technique used to record the separation profiles was that of Madsen and Latz (14) for their direct qualitative thin-layer chromatogram analysis. The 356-nm emission of a mercury-xenon lamp was employed for excitation and the emission monochromator was set at 532 nm. The slit program was 5-4-0.5-5-5 and the meter multiplier dial on the photomultiplier microphotometer was set at 0.01.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
1355
Table 111. Number of Fractions Obtained for the Separation of Commercial ABS, under Various Buffer Conditions in Aqueous Agarose Number of fractions Concn, M Buffer type 215 225 230 PH 0.025 Triethylamine 3 3 3 0.020 Acetic acid 10 1 1 2 0,050 Phosphate 8 1 1 2 0.025 Phosphate 8 2 2 2 I 0.050 Phosphate 1 1 2 I 0,025 Phosphate 1 1 1 7 0.050 Tris-HCI 2 2 2 0.050 Phosphate 6 1 1 2-3 0.025 Phosphate 6 1 1 2 5 0,025 Acetate 1 1-2 2 5 0,025 Phthalate 1 1 2 5 0.025 Phosphate 1 1 1 0.020 Citrate 5 2 2 2 5 0.025 Butanoate 0.025 Acetate 3 3 5 0.012 Trimethylamine HCI 3 0.012 Acetate 2 2 2 5 0.010 N(CH$CH&Br 0.025 Triethylamine 3 3 3 0,050 Acetic acid 5 0.025 Triethylamine 3 3 3 5 0,050 Butanoic acid 1 1 1 5 0.012 Acetate '
4-5
4 4
0.025 0,050 0,050 0,050
Tris
Acetic acid Acetate Phosphate
1
1
1 2
1
2
1 3 2
RESULTS AND DISCUSSION
Aqueous Agarose Gels. PRELIMINARY STUDIES. Early results indicated that the relative electrophoretic mobilities of surfactant types were dependent on the buffer used in the media, Under some conditions, certain surfactants had similar mobilities while under other conditions their mobilities were quite different. Higher ionic weight l-AS had, under certain experimental conditions, a greater mobility than lighter homologs. Commercial ABS and AES mixtures had mobilities greater than p-methylbenzenesulfonate (p-1-1-S), p-butylbenzenesulfonate (p-1-4-S), and p-heptylbenzenesulfonate (p-1-7-S) under most conditions. The order of relative migration rates of various surfactants tended to approach the order expected, on the basis of relative ionic weight, when they were run in lower pH media. All of this erratic behavior is due to the monomer-micelle equilibrium that exists in these surfactant solutions. The tendency to approach normal behavior in lower pH media is thought to be due to an increase in the monomer/micelle ratio. AS, ABS, and AES are salts of strong acids and any acid-base equilibrium effects would be negligible in weakly acidic (pH 3) media. This increase in the monomer/micelle ratio is probably due to the greater amount of diffusion that occurs in the less structured gels formed at this pH. These results indicate that the relative mobilities of monomers and micelles as well as the monomer/micelle ratio are affected by the media. 1356
-
1-ALKYLBENZENESULFONATES. The electrophoretic properties of the 1-ABS in aqueous agarose were investigated using the 2 0 . cell. ~ ~ Initial runs were made using 0.50 thick, 1.0% agarose gels at cooling temperatures 17 to 20 "C. The applied potential was usually 500 volts and the time was varied from 60 to 20 minutes. Malachite green or BaCll was used to detect the samples after migration. Runs were made using pH 7, 0.025M phosphate, pH 6, 0.0125M phosphate, and pH 9, 0.05M H3B03-NaOH-KC1 buffers, with the complete series of 1-ABS. The migration distances for p-1-16 to p-1-64 were always in the correct order and if the concentration of the samples was reduced from 50 mg/ml to 25 mg/ml, the order of migration was correct for p-1-1-S to p-1-8-S. The low solubility of 1-ABS homologs heavier than p-1-8-S, necessitated the use of saturated solutions, at 20-40 "C, of these samples. The electrophoresis of the heavier homologs resulted in precipitation at the starting point or narrow streaks from the starting point to various distances. Also p-1-8-S gave an elongated spot, whereas the spots were round for the smaller members of the series. In an effort to solubilize and avoid precipitation of the higher molecular weight 1-ABS, they were dissolved in solutions of lauryl sulfate or commercial ABS. The electrophoresis of these samples gave one major fraction and sometimes a minor secondary fraction. The secondary fraction was u shaped and usually appeared at the same distance for all of the higher members of the 1-ABS series. The secondary compact u shaped fraction in both the lauryl sulfate and commercial ABS solutions, was suspected of being precipitated during the run. This probably results from a decrease in the local concentration by diffusion, of the solubilizing lauryl sulfate or commercial ABS. When the concentration is decreased below the critical micelle concentration, the lauryl sulfate or ABS can no longer solubilize the 1-ABS in their micelles, thus the 1-ABS are precipitated. COMMERCIAL ABS MIXTURES.The commercial ABS mixtures, 215,225, and 230, were investigated using the 20-cm cell. Several runs were made under various experimental conditions in 1.O-mm, 1.O aqueous agarose gels at cooling temperatures of 25 to 27 "C. The ABS mixtures were 50 mg/ml in aqueous solution. The results of some of these runs are listed in Table 111. The number of fractions obtained from each sample and the relative migration rates of fractions from a particular sample were both dependent on the buffer used. The relative amounts of material found in these fractions were dependent on the buffer used. Also p-3-12-S usually separated into two fractions and the relative migration rate of p-3-12-S/ laurylsulfate was dependent on the buffer used. Laurylsulfate which was later found to contain 1-dodecylsulfate, 1-tetradecylsulfate, and 1-hexadecylsulfate always gave one spot under these conditions. Thus, separations obtained from surfactant mixtures containing micelles cannot be interpreted on the basis of ionic weight distribution. To gain more information about the monomer-micelle behavior, solutions of individual l-alkylsulfates were investigated. 1-ALKYLSULFATES. Electrophoresis was applied in the same manner as in the preceding study of the ABS. The electrophoresis of dodecylsulfate (1-10-s), undecylsulfate (1-11-s), and dodecylsulfate (1-124) at 50 mg/ml in pH 5 , 0.025M acetate and pH 5, 0.0125M citrate buffers produced two fractions, one more dense than the other. The smaller the alkylsulfate, the more material it had in the less dense fraction. In pH 5 , 0.050M acetic acid-0.025M triethylamine
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
buffer, the order of migration was 12 > 14 > 11 > 10. The same results were obtained using a pH 10, 0.020M acetic acid-0.030M triethylamine buffer. A series of runs were made using pH 3.5 and pH 4.5 acetate buffers ranging in concentration from 0.025M to 0,125M with a triethylamine concentration of 0.01M. The triethylamine was added in order to keep 1-14-s in solution. As the ionic strength was increased, the proportion of material in the dense fraction also increased. In general, the members of the 1-AS, depending upon concentration, ionic weight, and experimental conditions, appeared as a single compact spot, a diffuse spot, a leading compact spot with either diffuse tailing or a diffuse spot with a slower migration rate, and a spot with a compact center and diffuse outer ring. Diffuse areas are attributed to the presence of monomers whereas the compact spots result from high micelle concentration. These results clearly indicated the necessity for the elimination of micelles from all surfactant samples if a meaningful separation of surfactant mixtures was to be attained. It was thought that the presence of micelles could be eliminated by the addition of organic solvents to the gel composition and this prospect was investigated. Aqueous Organic Agarose Gels. PRELIMINARY STUDIES. The first organic solvent selected for study was dimethylformamide (DMF). Aqueous solutions of DMF, as high as SO%, form rigid transparent gels with agarose. The first results using D M F were an increase in diffusion, a decrease in mobility, and a decrease in detectability with the dye malachite green. However, these early results also showed that the relative migration rates of the 1-alkylsulfate series were, for the first time, in correct order. The undesirable effects caused by the addition of D M F were diminished by the use of pinacryptol yellow as the detecting agent, decreasing the concentration of the samples in order to decrease the rate of diffusion, and by increasing the electric field strength. A search of the literature produced the names of other organic compounds that had micelle-denaturizing effects on aqueous solutions of ionic surfactants (16-18). A study was made on the effects of several of these organic additives on the electrophoretic properties of a homologous series of 1-AS and 1-ABS. These studies were carried out using pH 4-5,0.025-0.0125M acetate buffered, 1.274, 1.0-mm thick agarose gels. The concentration of organic additives varied from 10 to 50%. The initial studies were made using the 20-mm cell, with cooling temperatures 17 to 20 "Cand an applied potential of 500 volts. The 1-ABS were soluble in aqueous 1-propanol which has micelle-denaturizing properties at high concentrations and micelle-stabilizing properties at low concentrations in aqueous solutions (17, 18). In an experiment using a 35% propanol gel, the effects of surfactant concentration on its monomermicelle composition can be clearly seen. p-1-124 was run at concentrations of 10, 5 , 2.5, 1.25, 0.62, and 0.31 mg/ml for a duration of 0.5 hour. The shape of the spot progressed from an elongated elliptical form (micelles present) at 10 mg/ml to a circular form (monomer) at 1.25 mg/ml and below. The near end of all the spots was the same distance from the origin, while the distance from the front of the spot to the origin decreased with decreasing concentration. (16) K. Shirahama and R. Matuura, Bull. Chem. SOC.Japan, 38, 373 (1965). (17) M. F. Emerson and A. Holtzer, J. Phys. Chem., 71, 3320 (1967). (18) K. Shirahama, M. Hayashi, and R. Matuura, Bull. Chem. SOC. Japan, 42, 1206 (1969).
If the results of running the I-ABS series, p-1-10-S to
p-1-14-S at 20 mg/ml in 20% propanol gel ale compared to the results obtained from a 35% propanol gel, the order of migration is completely reversed. The effectiveness of the elimination of micelles in various aqueous organic additive gels was thus evaluated by the spot shape and order of migration rate for the 1-ABS or 1-AS homologous series. The various organic additives used and found to form gels containing 1 . 2 z agarose were DMF, 1-propanol, p-dioxane, acetamide, cellosolve, ethyleneglycol, glycerol, 1,2-propanediol, and urea. From this study, it was concluded that the D M F and dioxane systems gave the best results. However, these results also indicated that a longer migration distance was needed for resolution of adjacent members of the ABS series. A cell with the same design as the DesagaBrinkmann cell, except with a cooling block that would accept a 35-cm plate, was built and put into service. When the run time was increased from 1.5 to 2.75 hours to obtain the maximum migration distance in the 35-cm cell, an undersirable characteristic, under the previous conditions, became a major problem. There had been some surface solvent transport in both the DMF-acetate and dioxane-acetate systems when using the 20-cm cell, but it did not interfere, to any extent, with the separation. However, the longer time period allowed the solvent, which travels from the anode toward the cathode, to intercept and pass the samples which are migrating toward the anode. A loss in definition of the sample spots was caused directly by diffusion of the sample into the free surface solvent, and indirectly by electric field disturbances. There was also a high resistance line, which moved from the anode toward the cathode, though not necessarily originating at the anode. The visible effects, caused by the encounter of a sample with a high resistance line, was to cause the spot to become elliptical with its major axis perpendicular to the direction of migration. This sometimes caused better resolution to be obtained for mixtures, but more often than not it also caused gel deterioration and its effects were not constant throughout the width of the gel. Dioxane gels were then run in pH 6 and 7, 0.010M phosphate buffer, which eliminated all surface solvent transport. However, the high resistance line still occurred but with less frequency. Another problem was that the gel between the cathode wick and starting holes became quite thin. This problem of apparent solvent loss was solved by placing a cellulose strip between the gel and the paper-gauze wicks so that all transport between the gel and wick passed through the cellulose strip. ~-ALKYLSULFATES. The separation of 1-alkylsulfates had been attained using l0-15% DMF, 1.0% agarose gels buffered at pH 4-5 with 0.0125M acetate in the 20-cm cell. The concentration of each alkylsulfate in the mixture was 1.25 mg/ml. In order to attain better resolution for mixtures of greater concentration, the 1-AS were run in the 35-cm cell using dioxane, 1.2% agarose gels, buffered at pH 6 with 0.01M phosphate. The results of two typical runs under these conditions are listed in Table IV and Table V. The results of this study indicate that 1-14-s, at a concentration of 5.0 mg/ml, usually tails in a gel of dioxane concentration less than or equal to 30%. If the concentration of 1-14-s is lowered to 2.5 mg/ml, tailing does not usually occur in a 20% dioxane gel. This tailing by 1-14-s is due to its insolubility in the lower per cent dioxane gels at the indicated temperature. All 1-AS lower than 1-14-s are quite soluble in aqueous or aqueous dioxane solutions and do not tail
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
1357
Table IV. Typical Migration Distances for 1-Alkylsulfates in 20 % Dioxane/Agarose Gels Run Time Was 2.25 Hours at a Potential of 1120 Volb and a Coaling Temperature of 18 "C with a Current of 55 mA Individual Concn, mg/ml Migration, cm standards in 5 0 x DMF Front Back 1-lo-s 5 .O 28.8 27.7 1-114 5.0 27.7 26.3 1-12-s 5 .O 26.5 25.1 1-14-s 5.0 23.3 21.8 Mixture 1-lM 1.25 29.0 28.1 1-114 1.25 27.5 26.5 1-12-s 1.25 25.9 24.9 1-14-s 1.25 24.0 22.9
I
Typical Migration Distances for 1-Alkylsulfates in 50 % Dioxane/Agarose Gels Run Time Was 2.0 Hours at a Potential of 1540 Volts and a Cooling Temperature of 18 "C with a Current of 46 mA Individual Concn, mg/ml Migration, cm. standards in 50% DMF Front Back 1-lo-s 2.5 ... 1-114 2.5 24.3 23 5 1-124 2.5 23.2 22.2 1-14-s 2.5 21.1 19.6 Mixture 1-10-s 1.25 ,.. ... 1-11-s 1.25 24.2 23.8 1-12-s 1.25 22.9 22.3 1-14-s 1.25 20.9 20.1
4
Table V.
L 2 7 2 5 23 21 IS 17 MIGRATION, CM Figure 1. Separation protiles of standard and commercial AS mixtures: (a) standard, 3 mg/ml/component; (b) standard, 5 mg/ml/component; (c) LS-1, 10 mg/ml; (d)LS-2,10 mg/ml 1. Decylsulfate 2. Undecylsulfate 3. Dodecylsulfate 4. Tetradecylsulfate 5. Hexadecylsulfate
Table VI. Typical Migration Distances for 1-Alkylbenzenesulfonate Mixtures in 50 Dioxane/Agarose Gels Run Time Was (a) 2.67 Hours (b) 2.83 Hours at a Potential of 1540 Volts and a Cooling Temperature of 18 O C with a Current of 46 mA Concn, w/ml
Sample Mixture (a) p-1-l0.S p-I-11-s p- 1-12-s Mixture p-1-12-s p-1-13-S p- 1-1 4 s Mixture (b) p-1-104 p-1-124 p - 1-1 4 s ~~
in 40% 1-propanol 0.50 0.50
Migration, cm Front Back
0.50
29.4 28.5 27.6
29.8 27.9 26.9
0.50 0.50 0.50
27.6 26.9 26.0
26.9 26.0 25.2
0.50 0.50
28.5 26.3 24.6
27.7 25.3 23.7
0.50
~~
even at much higher concentrations. All evidence indicates that 1-14-s, at a concentration of 5.0 mg/ml was completely in monomer form during its migration in a 30% dioxane gel. The results also indicate that 1-12-s and lower members of the series, at 5.0 mg/ml concentration, are in monomer form in gels containing only 10 % dioxane. There was no problem in visualizing the spot given by a 1.25 mg/ml sample of 1-10-s in a 20% dioxane gel. However, for dioxane concentration between 25 and 50%, 1-10-s, at 1.25 mg/ml was not usually detected. The solubility of the 1-10-s pinacryptol yellow adduct was also a problem in the DMF systems. The only solution so far is to vary the dioxane concentration for the particular needs of the sample. However, this does not detract from the good separations obtained by this system. A gel of 40-50% dioxane concentration should probably be used when working with samples 1358
between 5-10 mg/ml, especially ones containing a large percentage of the higher I-AS. Table VI lists some typical ~-ALKYLBENZENE~ULFONATES. results for the separation of 1-ABS mixtures obtained in pH 6, 0.01M phosphate, 50% dioxane, 1.2%, 1.25-mm thick agarose gels. The resolution obtained for the mixtures was quite adequate for qualitative comparisons between mixture and standards. However, for any intended quantitative use, better resolution would be required for mixtures containing p-1-13-S, p-1-14-S, and p-1-153. The absolute differences in migration distances for members of both the ABS and AS series are dependent only on the distance of migration. Their relative migration rates have not changed under the various running conditions, for which it was assumed all species were in monomer form. Therefore, considering a set distance of migration, resolution can best be improved by reduction in spot size. Spot size is dependent on diffusion and on the original size of sample application. Diffusion is dependent, among other factors, on concentration and time, and so the effects of a reduction in both of these were studied. While the higher 1-ABS can be detected at concentrations lower than 0.50 mg/ml, the goal of this study is to separate and detect the more soluble ABS isomers. A distinction should be made between real separations and apparent separations. Because of the solubilities of the pinacryptol yellow adducts, the precipitated zones may not necessarily correspond to the total sample zone. Therefore, reducing the concentrations of the 1-ABS below the detection limit
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
Figure 2. Separation profiles of standard 1-ABS and commercial ABS mixtures : (a) 215, 5 mg/ml; (b) 225, 5 mg/ml; (c) SI,0.5 mg/ml component; (6)52, 0.5 mg/ml/component; ( e ) 230,5 mg/ml 1. 2. 3. 4. 5.
p-*-lo-s p-*-11-s p-*-12-s p-*-13-S p-*-14-s
* All isomers of a speclfic
>I-
v)
z W
I-
z W
>
n"
a
0
L Ab
hc
2
d
Ididi
a
5
4
e
l-
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of the more soluble ABS isomers would not contribute to this final goal. By decreasing the buffer concentration to 0.005M phosphate and increasing the voltage while keeping joule heat output near constant, the time for a 28-cm migration for p-1-10-S was reduced to 2.0 hours. This reduction in time did not result in a significant decrease in diffusion and thus no improvement in resolution. I n fact, the spots for the higher 1-ABS homologs tended to be slightly elongated, thus resolution for mixtures of the higher homologs was decreased under these conditions. Another approach to gain better resolution was to reduce the size of the starting holes from 1.5-mm to 1.O-mm diameter, but this led to no significant reduction in spot size or resolution, The gel thickness was increased slightly while using the same amount of sample. This study was inconclusive because just a few runs indicated that the cooling process was not sufficient for these systems. Separation Profiles of Commercial and Standard Mixtures. All the following separations were obtained using the 50% dioxane gel described under Procedure. The use of this particular gel, cooling temperature of 18 "C,and applied potential of 1540 volts will henceforth be referred to in this paper as standard conditions. ALKYLSULFATES. Mixtures of 1-10-s, 1-11-s, 1-12-s, and 1-14-s have been separated in 2.0 hours under standard conditions. The migration distance of 1-12-s in 2.0 hours under these conditions is approximately 24 cm. The separation profiles, Figure 1, indicate good resolution for this standard mixture when each component is at a concentration of 5.0 mg/ml. However, the indication of resolution at this concentration is misleading due to the solubility of the pinacryptol yellow adduct of the lower members of the series. As previously mentioned, a distinction must be made between
zz 20
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3 0 2 8 20 24
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Figure 3. Separation profiles of standard 1-ABS and commercial ABS mixtures: (a) Ucane 11, 5 mg/ml; (6) Ucane 12, 5 mg/ml; (c) SI, 0.5 mg/ml/ component; (d) Si, 0.5 mg/ml/component; (e) Ucane 13, 5 mg/ml; (f) p-3-12-S, 2.5 mg/ml 1. p-*-lo-s 2. p-*-11-s 3. p-*-12-s 4. p-*-13-S 5. p-* -14-5
* See caption to Figure 2
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MIGRATION, CM
Figure 4. Separation profiles of standard ABS: (a) pl-12-S, 0.5 mg/ ml; (6) p-1-1043, p-l-12-S, and p 1-1443, 0.5 mg/ ml/component; (c) p-3-1243, 2.5 mg/ ml 1. 2. 3. 4.
p-1-104 p-1-12-s p-1-14-S p-3-12-S
apparent and real separations. A more critical evaluation would probably indicate that a 2.0 mg/ml concentration for each component is the maximum concentration for this particular mixture to obtain complete resolution of all its components. The separation profiles of lauryl sulfate samples (LS), when compared to the profile of the standard mixture, Figure 1, show the presence of dodecyl-, tetradecyl-, and hexadecylsulfates. No fraction corresponding to decylsulfate has ever been detected even under conditions that were more favorable for its detection. However, it may be present as a minor component. These profiles of lauryl sulfate show the high degree of resolution that may be attained for derivatives of natural products. The sodium sulfate derivative of 7-ethyl-2-methyl-4undecanol was also examined under these conditions and migrated the same distance as 1-14-s as expected. The branched four-substituted isomer is, of course, more soluble than the straight chain primary isomer. It cannot be detected
ANALYTICAL CHEMISTRY, VOL. 43, NO. 1 1 , SEPTEMBER 1971
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1.46
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LOG IONIC WEIGHT
Figure 5. Log migration distance us. log ionic weight for the 1-ABS separation profile shown in Figure 4 below the 5-pg level whereas 1-14-s can be easily detected at the 1.O-pg level using present techniques. ALKYLBENZENESULFONATES. Six commercial mixtures of ABS have been investigated using this method. They were run under standard conditions for 2.75 hours in which time (p-1-12-S) migrated a distance of 27 cm. The components of these mixtures are identified by comparing their separation profiles with those of standard mixtures, Figures 2 and 3. The standard mixture, SI, contains (p-1-10-S), (p-1-11-S), and (p-1-129, each at a concentration of 0.50 mg/ml in 40% 1-propanol. The standard mixture, S2, contains (p-1-12-S), (p-1-13-2), and (p-1-14-S), each at a concentration of 0.50 mg/ml in 4 0 z 1-propanol. A sample of (p-3-12-S) was also run as an additional standard. The commercial mixtures were run at concentrations of 5.0 mg/ml as the best compromise between resolution of major components and the detection of minor components. The separation profiles, Figures 2 and 3, indicate the resolution that was attained for both the synthetic standards and commercial mixtures. The leading spots in these separations were very close to the anode wick and thus the profiles do not give the natural separation distance between the first and second spots. Figure 4 is the separation profile of a mixture of p-1-10-S, p-1-12-S, and p-1-144 obtained under standard conditions, but for a shorter time period (2 hr, 35 min). If it is assumed that the migration distance (4,for a given run, is inversely proportional to the ionic weight (w),then a plot of log d us. log w should yield a straight time. Figure 5 is such a plot for the separation profile of Figure 4. These results show that while p-1-134 and p-1-14-S can be distinguished from one another, a greater distance of migration is needed for complete resolution. All experimental results indicate that there is no difference in mobility between isomers of the same ionic weight. However, there is a large difference in solubility between the 1-ABS and other isomers. The 1-ABS-pinacryptol yellow adduct fluoresces yellow while p-3-124 and the commercial ABS adducts fluoresce orange-yellow. Thus the detection limit for the 1-ABS isomer is lower than for the other isomers, Figure 4,a and c. ALKY LETHOXYSULFATES A N D ALKY LARY LETHOXY SULFATES. The commercial AES mixtures of 25-s-3A, 45-s-3A, 15-s-3S, and 15-s-5A were examined using this method under standard conditions. The samples were at a concentration of 20 1360
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Figure 6. Separation profiles of commercial AES and AAES mixtures, AS and ABS standards: (a) p-3-12-S, 2.5 mg/ml; (b) Alipal CO 436, 20 mg/ml; (c) 45-s-3A, 20 mg/ml; (d) 25-s-3A, 20 mg/ml; (e) LS-2, 10 mg/ml; (f)15-s-3S, 20 mg/ml mg/ml and run times were 2.0 to 2.5 hours. Electrophoresis of these mixtures gave eight to twelve fractions for 25-s-3A, seven fractions for 15-s-3S, and six fractions for 45-2-3A. Although a few very light spots could be seen for 15-s-5A, they were of such poor definition that no data could be recorded for them. This is due to the solubility of their pinacryptol yellow adduct. The products, 25-s-3A, 45-s-3A, and 15-s-3s are all similar in that they are derived from primary linear alcohols and are suppose to contain three moles of ethylene oxide adduct. 15-s-5A is derived from linear secondary alcohols and is suppose to contain five molecules of ethylene oxide adduct. The nature of the mixture is described by the code name of the sample. The first number of the code name 25-s-3A denotes that the product was derived from C12-1~ alcohols, s denotes sulfate, 3 denotes number of ethylene oxide molecules added to the alkyl chain, and A denotes the ammonium salt while an S in this position denotes the sodium salt. The separation profiles of these products, shown in Figures 6 , 7 , and 8 are difficult to interpret without standard mixtures of these compounds and/or knowledge of the distribution of the starting materials. A standard alkylsulfate mixture and a standard 1-ABS mixture were run along with these compounds to identify them by an ionic weight/mobility comparison. However, there are a number of combinations of ethylene oxide groups and starting alcohols which give products of ionic weight within three atomic units of each
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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Pip,8. Separation of commercial alkylethoxysulfate etmes in a 50% dioxaoe/agarose gel. Run time was 2.33 hours at a potential of 1540 volts and a cooling temperature of18 O C with a current of 45 mA
M I G R A T I O N . CM
Figure 7. Sepvatioo proruaS of eomnercial AES and AAES mixtures, AS and ABS standards: (a) tetradeeylsulfate, 2.5 mg/ml; (6)45-~-3A,20 mg/ml; (c) p3-12s,2.5 mg/ ml; (d) Alipal CO 436, 20 mg/ml; (e) pl-1023, pl-12-S, and pl-14.5, 0.50 mg/ml/cnmponent other. This, along with the fact that a n alkylethoxysulfate may not have the same mobility as an alkylsulfate or alkylbemnesulfonate ion with the same weight, would indicate that this method of comparison by itself is unsatisfsctory. However, the separations obtained from these mixtures are quite reproducible in respect to the separation itself and in comparison to the mobilities of other compounds. Thus it is believed that positive identification can he made by the use of proper standards. CONUUSION A great number of runs were made in the development of the present system using several different buffers at various pH, several organic additives, and many variations in experimental conditions. An improvement in resolution without a radical cbauge i n cell design is not expected. In theory, resolution could be improved by extending the length of the migration path. This could be accomplished by building a cell of the same design but slightly longer. However, there are indications that many of the factors in the present system are already beyond tbeu maximum aciency. The main problems in the present system are the occasional Occurrence of the high resistance line, which can cause minor to total disruption of the separation, and the difficulty of detection of the lighter surfactants at low concentrations with piaanyptol yellow.
The high resistance line is caused by the precipitation of buffer salts or assxiation of buffer ions. The cause for the progresive movement of this region toward the cathode has not been thoroughly investigated. However, the following ohsemtion has been made. An acidic front develops from the anode, due to the low buffer capacity, which, as it moves toward the cathode, redissolves the precipitated salts. It is thought that the initial build up of salts takes place in the region where the anode wick makes contact with the gel. However, it is not certain whether the movement toward the cathode is caused by the development of new regio- or by the travel of a single region. It is felt that this problem can be eliminated with the proper cell design. The analysis of a homologous series of ionic surface active agents by electrophoresis in aqueousdrganic solvent agarose gels bas advantages over paper and thin-layer chromatographic methods. Run times are considerably shorter and mobility is strictly dependent on ionic weight. The sample can also be extracted from the gel, as in the former methods, for quantitative work. The advantage of an electrophoretic method over pyrolytic-gas chromatography methods is that the molecules remain unchanged, which leads to a simple separation profile, based on ionic weight, as opposed to the very complicated distributions obtained from the latter method when isomers are present. The authors believe that the method presented in this paper can be used to advantage in the qualitative analysis of ionic surface active agents and other ionic substances. This is particularly true in the instances where electrophoretic methods fail because of micelle formation in aqueous solutions. With proper calibration, the area under the separation proliles can be used for quantitative estimates of the components and more accurate quantitative results can he obtained by applying established methods to the separated
components. Future studies will he wnmed with the quantitative aspects of the present system and the design of a new d, which hopefully will eliminate the undesirable characteristics of the present system and extend the migration path.
RECEIVEDfor renew February 17, 1971. Accepted May 25, 1971.
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