Identification and separation of cationic and non-ionic surfactants by reversed phase thin layer chromatography

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1 Indian Journal of Chemical Technology Vol. 5, May 2008, pp Identification and separation of cationic and non-ionic surfactants by reversed phase thin layer chromatography Ali Mohammad* & Rubi Gupta Analytical Research Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh , India Received 9 July 2007; revised 4 January 2008 Reversed phase thin layer chromatography (RPTLC) of non-ionic and cationic surfactants was performed on silica gel layers impregnated with 5% paraffin oil, silicone oil and tributyl phosphate as stationary phases using water and organic polar solvents as mobile phases. In all, four stationary phases and twelve mobile phases were used to examine the mobility pattern and to find out best TLC system for separation of surfactants from their mixtures. The TLC system comprising of 5% paraffin oil impregnated silica gel layers as stationary phase and pure methanol as mobile phase was found most favourable for achieving separation of non-ionic surfactants (Tx-00, Brij-35 and Tween-20) from cationic surfactants (CPC, CTAB and TTAB). The effects of loading amount of analyte and metal cations impurities in the sample have been examined on mutual separation of Tx-00 and TTAB. The results obtained with methanol were compared with those obtained with ethanol and butanol. The mobility of surfactants was found to increase with the increase of polarity of the alcohol used as mobile phase. Keywords: Reversed phase TLC, Surfactants, Identification, Separation Amphiphilic (bifunctional) compounds with a hydrophobic and hydrophilic part in the molecule are termed surfactant. These are interface active substances and often called the heart of washing powder or cleaner. Because of their favourable physicochemical characteristics, they are used in agrochemical formulation 2, pharmaceuticals 3, industrial 4 and house-hold products 5. Furthermore, surfactants have been successfully employed in biological activity as they readily bind to peptides 6 and influence the enzymes 7. Surfactants have been found useful in the removal of heavy metal from waste streams 8. Besides the beneficial effects, surfactants also show toxic behaviour 9. Several analytical techniques 0-3 have been used for quantitative analysis of surfactants. Thin layer chromatography (TLC) 4,5 being a versatile technique is more suitable for routine qualitative analysis of large number of surfactants. In addition to normal phase TLC, many workers have used reversed phase chromatographic methods for the analysis of surfactants 6-8. Reversed phase thin layer chromatography (RPTLC) of surfactants 9-2 using undecane, silicone oil, cholestrol as impregnants in combination with aqueous methanol or methanol plus ethyl acetate as mobile phases have been more commonly used for analysis of surfactants. Paraffin oil, one of the most versatile extractants has received considerable attention in analytical separation chemistry. Cserhati 22 has utilized silica gel layers impregnated with paraffin oil to examine the degree of lipophilicity of some mono amine oxidase inhibitors. Dansylated amino acids have also been analysed 23. Silica gel 60 G, F 254 plates impregnated with 2.5% paraffin oil have been used to examine the chromatographic behaviour of s-triazine derivatives 24. Kieselguhr layers impregnated with 0-5% paraffin oil have been applied for identification and separation of organic compounds Alumina layers impregnated with 5% paraffin oil in the hexane have been used to investigate the interaction of surfactants with peptides using methanol-water mixture (0 and 90% v/v) as mobile phase 28. Thus, there is only one report on the use of paraffin oil as impregnant in the analysis of surfactants. In view of excellent chromatographic performance of methanol as eluent in reversed phase TLC, paraffin oil as impregnant and little reported work on reversed phase TLC of surfactant, it was decided to utilize paraffin oil impregnated silica gel layers as reversed phase medium for the separation of surfactants using methanol as eluent. Cationic surfactants are widely

2 272 INDIAN J. CHEM. TECHNOL., MAY 2008 used as fabric softners. The presence of non-ionic surfactants influence their performance and hence the removal of nonionic surfactants from cationic surfactants before their use as fabric softners is important. Experimental Procedure Apparatus A TLC apparatus (Toshniwal, India) was used to prepare thin layers (0.25 mm) of various adsorbents on 20 3 cm glass plates. Glass jars (29 6 cm) were used for the development of TLC plates. Chemicals and reagents Silica gel G, acetone and acetonitrile were obtained from Merck, India. Methanol, ethanol, butanol, formic acid and ethylmethyl ketone were obtained from Qualigens, India. Tetrahydrofuran, sodium formate, ethyl acetate, paraffin oil, silicone oil, diethyl ether, dimethyl sulfuoxide (DMSO) and petroleum ether were obtained from CDH, India. Tributyl phosphate (TBP) was obtained from Indian Drugs and Pharmaceuticals Ltd. The cationic surfactants such as cetylpyridinium chloride, CPC and N-cetyl N,N,N-trimethyl ammonium bromide, CTAB both were obtained from CDH, India and tetradecyl trimethyl ammonium bromide, TTAB obtained from Merck, India. Nonionic surfactants such as isooctyl phenoxy polyethoxy ethanol, Tx-00 and polyoxyethylene dodecyl ether, Brij-35 were obtained from Loba Chemie, India whereas polyoxyethylene sorbitan monolaurate, Tween-20, polyoxyethylene sorbitan monopalmitate, Tween-40 and polyoxyethylene sorbitan monostearate, Tween-60 were received from CDH, India. The test solutions (%) of all surfactants were prepared in methanol. Detection Dragendroffs reagent was prepared by mixing two solutions: A (i) a solution of bismuth subnitrate (BiONO 3.H 2 O,7 g) in acetic acid (20 ml), diluted to 00 ml with water and (ii) a solution of potassium iodide (65 g) in water (200 ml). These solutions were transferred to L flask, acetic acid (200 ml) was added, and the solution was diluted to one liter with water. Solution B was prepared by dissolving barium chloride dihydrate (BaCl 2.2H 2 O, 290 g) in water ( L). Solution A and B were mixed in the ratio 2:. A glass sprayer was used to apply the reagent to plates to detect surfactants by observing orange coloured spots. Preparation of TLC Plates (i) Silica gel thin-layer plates TLC plates were prepared by mixing silica gel G with double distilled water in a :3 ratio. The resultant slurry was mechanically shaken for 5 min, then it was coated onto glass plates with the help of a TLC applicator to give a layer of mm thickness. The plates were first air dried at room temperature and then activated at 00 C by heating in an electrically controlled oven for h. The activated plates were stored in closed chamber at room temperature until used. (ii) Paraffin oil impregnated TLC plates Two methods are adopted for the preparation of impregnated silica gel plates (i) precoating method and (ii) post coating method. In this paper the post coating method has been used. In this method the activated silica gel plates were impregnated with desired concentration of paraffin oil (5-20%, v/v) in diethyl ether by dipping silica gel plates in solution of impregnant for a specific time period followed by drying of the plates at room temperature (30 C). This post coating method was also found suitable for the preparation of tributyl phosphate (in acetone) and silicone oil (in petroleum ether) impregnated TLC plates. Method The activated plates were marked with two horizontal lines 2 and 2 cm from the base. The surfactant solutions µl were spotted on TLC plates with micropipette (Tripette, Germany). The spots were allowed to air dry and the plates were developed in chosen mobile phase by one-dimensional ascending technique in glass jars. The solvent ascent was fixed to 0 cm from the point of application in all cases. After development, the TLC plates were dried at room temperature and the spots of surfactants were detected using Dragendroffs reagent. The R F values [R F = (R L + R T ) /2] were calculated from R L (R F of leading front) and R T (R F of trailing front) values of detected spots on TLC plate. The reproducibility (or precision) of R F values was checked by determining the R F values of the same sample by the same analyst on different days under identical experimental conditions, using the same apparatus. The variation in R F values differs by a factor of ± (i.e. ± 0%) from the average value, indicating a good reproducibility.

3 MOHAMMAD & GUPTA: IDENTIFICATION AND SEPARATION OF CATIONIC AND NON-IONIC SURFACTANTS 273 To study the effect of the presence of metal ions ( M) as impurities on the separation of TTAB and Tx-00, μl each of TTAB and Tx-00 standard test solution was sported on TLC plate (S 2 ) followed by spotting of the metal ions ( μl), the plates were developed with M 3, detected and R F values of the separated surfactants were calculated. Limits of detection of some surfactants were determined by loading different amounts of surfactants on the TLC plates, developing the plates and detecting the spots. The method was repeated with successive lowering of amount of surfactants until no spot was detected. The lowest detectable amount of surfactants on TLC plates was taken as the limit of detection. Results and Discussion The chromatographic systems, mobile and stationary phases used in present study are listed in Table. Starting with double distilled water (M ), acetone (M 2 ) and methanol (M 3 ) as mobile phases, the mobility pattern of surfactants (cationic and nonionic) on plain (or unimpregnated) silica gel layers was examined. As evident from the data listed in Table 2, all surfactants remain near the point of application (R F =0.0) on silica gel layer (S ) developed with water (M ). In contrast, all surfactants migrate with solvent front showing high mobility (R F = ) when pure acetone (M 2 ) was used as mobile phase. On using methanol (M 3 ) as mobile phase, Tx-00 and Brij-35 showed high mobility (R F Table The solvent and adsorbent systems used as mobile and stationary phases respectively Solvent systems Adsorbent System Symbol Composition Symbol Composition M Double distilled water S Silica gel G M 2 Acetone S 2 Silica gel G impregnated with 5% paraffin oil in diethyl ether M 3 Methanol M 4 M Formic acid S 3 Silica gel G impregnated with 5% silicone oil in petroleum ether M 5 M Sodium formate M 6 Ethyl acetate S 4 Silica gel G impregnated with 5% tributyl phosphate (TBP) in acetone M 7 Acetonitrile M 8 Tetrahydrofuran (THF) M 9 Dimethyl sulphoxide (DMSO) M 0 Ethyl methyl ketone M Ethanol Butanol M 2 Table 2 R F values of surfactants obtained on silica gel layer impregnated with 5% paraffin oil (stationary phase, S 2 ) and developed with various mobile phases (M - M 0 ) Surfactants R F Values M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 0 Tx (0.95)* 0.65 (0.85)* (T) 0.68 Brij (0.92) 0.62 (0.80) ND (T) 0.45(T) 0.70 Tween (0.94) 0.58 (0.72)(T) Tween (0.95) 0.45(T) (0.62)(T) Tween (0.94) 0.48(T) (0.82)(T) CPC 0.78 (0.90) (0.72)(T) CTAB 0.79 (0.92) 0.08 (0.75)(T) TTAB 0.80 (0.92) 0.07 (T) All the surfactants remain near the point of application (R F = 0.0) on unimpregnated silica gel (S ) and impregnated silica gel G (S 2 ) layers developed with water (M ). *R F values recorded in bracket refer to the values obtained on unimpregnated silica gel layer. ND refers to not detected T refers to tailed spots (R L -R T 0.30)

4 274 INDIAN J. CHEM. TECHNOL., MAY 2008 around 0.85) whereas other nonionic surfactants (Tween-20, Tween-40 and Tween-60) and cationic surfactants (CPC, CTAB and TTAB) produced diffused and tailed spots (R L -R T 0.30 where R L and R T are the R F values of the leading and trailing ends of the spots). Since plain silica gel layers could not produce satisfactory results with M -M 3, it was decided to utilize silica gel layers impregnated with 5-20% paraffin oil as stationary phase. The chromatography of surfactants was performed on silica gel layers impregnated with 5-20% paraffin oil using M -M 3 as mobile phases. With M and M 2 as mobile phases, marginal changes in mobility of surfactants were observed. In case of M 3, surfactants showed modified mobility offering their separation possibilities on 5% paraffin oil impregnated silica gel layers (S 2 ). The higher degree of paraffin oil (5-20%) was found unsuitable because the chromatography with silica plates impregnated at high degree of paraffin oil causes several problems including increase in development time, poor detection, lesser spot compactness and difficulty in drying of plates. With the increase in concentration of paraffin oil as impregnant there was a slight decrease in R F values. Furthermore, badly tailed spots for Tween 20, 40 or 60 and Brij-35 were observed on silica gel layers impregnated with 20% paraffin oil. In view of spot compactness, detection clarity, lesser development time and separation possibilities, a reversed phase TLC system comprising of silica gel layers impregnated with 5% paraffin oil as stationary phase (S 2 ) and methanol (M 3 ) as mobile phase was selected as most favourable TLC system. To find out some useful mobile phases, surfactants were also chromatographed on S 2 using other polar organic solvents as mobile phases (M 4 -M 0 ) and the results are listed in Table 2. All surfactants show identical mobility in M 4 -M 8 mobile phase systems and remain near the point of application. Thus, there was no separation possibility of surfactants with M 4 -M 8. With M 9 tailed spots for nonionic surfactants and low R F for cationic surfactants were noticed. However, all surfactants gave mid R F with M 0. When methanol (M 3 ) was replaced by ethanol (M ) or butanol (M 2 ), the mobility (in terms of R F value) of surfactants on S 2 was found to decrease in the following order: R F (MeOH) > R F (EtOH) > R F (BuOH) i.e. in the decreasing order of polarity of mobile phases (Fig. ). Thus, The TLC system including S 2 as stationary phase and M 3 as mobile phase was capable for separation of: (a) Tx-00 from CPC, CTAB and TTAB, and (b) Brij-35 from CPC, CTAB and TTAB. In order to understand the nature of the impregnant, silica gel layers were impregnated with 5% silicone oil (S 3 ) and tributyl phosphate (S 4 ). The results obtained on S 3 and S 4 have been compared with those realized on S 2 using M 3 as mobile phase (Fig. 2). There was significant reduction in mobility (or R F value) of all nonionic surfactants on tributyl phosphate (S 4 ) impregnated layer compared to the mobility on silicone oil (S 3 ) and paraffin oil (S 2 ) impregnated layers. Interestingly, cationic surfactants show almost identical behaviour and remain near the point of application on S 2 -S 4. Limit of detection of surfactants on silica gel layer (S ) and 5% Paraffin oil impegranted silica gel layer (S 2 ) developed with mobile phases (M -M 0 ) are presented in Table 3. It was observed that µg Tx-00 can easily be separated from 0.7 mg TTAB whereas µg TTAB can easily be separated from 0.2 mg of Tx-00. Thus, microgram quantities of one surfactant can be Fig. Plot of R F values versus surfactants Fig. 2 Plot of R F values versus surfactants

5 MOHAMMAD & GUPTA: IDENTIFICATION AND SEPARATION OF CATIONIC AND NON-IONIC SURFACTANTS 275 Table 3 Limit of detection of surfactants on silica gel layer (S ) and 5% paraffin oil impregnated silica gel layer (S 2 ) developed with mobile phases (M -M 0 ) Surfactants Limit of detection (μg) M M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 0 Tx-00 * Brij-35 Tween-20 Tween-40 Tween-60 CPC () CTAB () TTAB () () () () ND ND () () () () () () () () () () () () () () () () () () () () () () () () (0.0) (0.0) ND refers to not detected *μg values recorded in bracket refer to the values obtained on 5% paraffin oil impregnated silica gel layer Table 4 Experimentally achieved separations of surfactants on silica gel impregnated with 5% paraffin oil (S 2 ) layers developed with mobile phase (M 3 ) Surfactants Mixture of cationic (CPC, CTAB and TTAB) with nonionic surfactants. Mixture of nonionic surfactants (Tx-00, Brij-35 and Tween-20) with cationic surfactants Separations (R F Value) CPC, CTAB, and TTAB (0.08) Tx-00 (0.70) CPC, CTAB, and TTAB (0.08) Brij-35 (0.47) CPC, CTAB, and TTAB (0.08) Tween-20 (0.52) Tx-00, Brij-35 and Tween-20 (0.55) CPC () Tx-00, Brij-35 and Tween-20 (0.55) CTAB (0.08) Tx-00, Brij-35 and Tween-20 (0.55) TTAB (0.07) Tx-00 (0.65) TTAB (0.07) Tx-00 (0.65) CTAB and CPC () Brij-35 (0.62) TTAB (0.08) Tween-20 CPC, TTAB () successfully separated from milligram amounts of other surfactant by use of the proposed TLC systems. Table 4 summarized the experimentally achieved separations of surfactants. Each cationic surfactant individually separated from mixture of non-ionic surfactants and vice versa. From the data listed in Table 5, it is evident that mutual separation of TTAB and Tx-00 from their Table 5 Separation of TTAB from Tx-00 on silica layers impregnated with 5% paraffin oil stationary phase (S 2 ) and methanol (M 3 ) mobile phase in the presence of metal cations as impurities. Cations R F Values of separated spots TTAB Tx-00 Ni Cd Ca Pb Zn Fe (T) Cr T = Refers to tailed spots (R L R T 0.30) mixture is possible in the presence of all metal ions (Ni 2+, Cd 2+, Ca 2+, Pb 2+, Zn 2+, Cr 6+ ) except Fe 3+. The Fe 3+ interferes in the separation as it causes tailing in Tx-00 probably due to certain specific interaction. Conclusion The most favourable chromatographed system for separation of non-ionic surfactants (Tx-00, Brij-35, Tween-20) from cationic surfactants (CPC, CTAB, TTAB) was 5% paraffin oil impregnated silica gel layer as stationary phase and pure methanol as mobile phase. The mobilities of surfactants were influenced by the polarity of alcohols. The microgram quantities of cationic surfactant can be successfully separated

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