Ionic liquid as separation enhancer in thin-layer chromatography of biosurfactants: mutual separation of sodium cholate, sodium deoxycholate and sodium taurocholate
© Mohammad and Mobin; licensee Springer. 2015
Received: 16 February 2015
Accepted: 27 April 2015
Published: 15 May 2015
Physiological biosurfactants plays an important role in digestion of lipids and cholesterol also emulsifies fat-soluble vitamins.
A new green thin-layer chromatographic system comprising ionic liquid (1-methylimidazolium chloride) impregnated silica gel G and 2-methyl tetrahydrofuran as stationary and mobile phases was found to be most suitable for the separation of ternary mixture of biosurfactants (sodium cholate, sodium deoxycholate and sodium taurocholate). The surface structure and chemical composition of silica gel G modified by impregnation with 1-methylimidazolium chloride were examined with the aid of scanning electron microscopy and energy-dispersive X-ray spectrophotometry, respectively.
Compared to plain silica gel, enhanced separation efficiency of ionic liquid impregnated silica gel was observed for the resolution of biosurfactants from their mixture. Chromatographic parameters such as ΔR F, separation factor (α) and resolution (R S) for the separation were calculated. Effect of foreign substances (metal cations, inorganic anions, vitamins, amino acids and non-ionic surfactants as impurities) on the separation of surfactants was also examined. Effects of concentration level of 1-methylimidazolium chloride as impregnant and its substitution by other ionic liquids (1,2,3-trimethylimidazoliummethyl sulphate, 1-ethyl-3-methylimidazolium tetrafluoroborate) were also studied to decide the optimum experimental conditions for better separation possibilities. The limits of detection of sodium cholate, sodium deoxycholate and sodium taurocholate were calculated.
A new green thin-layer chromatographic system comprising ionic liquid (1-methylimidazolium chloride) impregnated silica gel G and 2-methyl tetrahydrofuran as stationary and mobile phases provide the most suitable environment for the separation of ternary mixture of biosurfactants.
KeywordThin-layer chromatography Ionic liquid Biosurfactants SEM EDX
Bile salts commonly occurring as sodium salts of bile acid and produced in the liver of mammals are known as primary bile acids, and those produced by the bacteria present in the colon are called secondary bile acids. Because of micelles’ forming properties, bile salts [sodium cholate (SC), sodium deoxycholate (SDC) and sodium taurocholate (STC)] have been popular as physiological biosurfactants (Santhanalakshmi et al. 2001; Matsuoka and Moroni 2002; Hofmann and Mysels 1987; Selvam et al. 2009). These biosurfactants play an important role in (a) digestion of lipids and cholesterol, (b) emulsifying fat-soluble vitamins to enable their absorption in the intestine and (c) excretion of bilirubin. Biosurfactants have found use in medicines and cosmetics as emulsifying and dispersing agents (Santhanalakshmi et al. 2001; Selvam et al. 2009). SC and SDC being water-soluble mild ionic detergents are used for the isolation of membrane protein and lipids. In affinity chromatography SC is used for preventing non-specific binding. It is also used in cell culture media supplements Sodium Cholate, Pierce Biotechnology, USA. SDC is used as a non-surgical cosmetic medicinal treatment in mesotherapy injection and acts as a photo-resistant component in microlithography (Nanda 2011; Matarasso and Pfeifer 2009; Kim et al. 2000). Thus, analysis of these biosurfactants has been important because of their physiological importance.
Various analytical methods have been used in separation science, but thin-layer chromatography (TLC) is the most popular analytical tool used for separation because it is simple, rapid, versatile and cost-effective (Mohammad and Hina 2005; Mohammad and Bhawani 2008; Mohammad et al. 2010; Mohammad et al. 2013). Several inorganic, organic and aqueous-organic mobile phases and different stationary phases have been used in TLC analysis of surfactants (Mohammad and Agrawal 2001; Mohammad et al. 2002; Touchstone 1992; Gocan 2002). Most of mixed aqueous-organic and organic solvents previously used in chromatographic analysis need to be replaced by environmentally benign solvent systems in order to reduce their adverse impact on the environment.
Our aim of the present study was to achieve important separations of biosurfactants making TLC green by using environmental friendly mobile phase and silica gel modified with ionic liquid as stationary phase. Ionic liquids (ILs) are organic compounds, which exist in liquid form at room temperature, and have received much attention for their use in chemical industries due to various salient features such as good thermal stability, non-volatility and non-generating harmful volatile organic compounds (VOCs) as products. ILs provide good medium to solubilise H2, O2, CO and CO2 gases and easily interact with other compounds via hydrogen bonding and electrostatic, dipolar, dispersive, pi-pi and n-pi bonding (Mallakpour and Dinari 2012).
In the present study, 1-methylimidazolium chloride ionic liquid is introduced in silica gel stationary phase which modifies the surface structure and provides better resolution of biosurfactants (SC, SDC and STC) in the presence of 2-methyltetrahydrofuran, a green alternative solvent for tetrahydrofuran as mobile phase. The use of green solvents in mobile as well as in stationary phases is a novel approach to develop environmental friendly green TLC method. It fulfils the growing demand of making analytical process greener. The proposed method was successful to identify SDC in formulated sample.
All experiments were performed at 25 ± 2°C.
A TLC applicator was used for coating silica gel on 20 × 3.2 cm glass plates and chromatography was performed in 24 × 6 cm glass jars. A glass sprayer was used to spray reagent on the plates to locate the position of the spot of the analyte.
Chemicals and reagents
Silica gel G (Thermo Fisher Scientific, Mumbai, India), alumina, kieselguhr, 2-methyltetrahydrofuran (CDH, New Delhi, India) and 1-methylimidazolium chloride, 1,2,3-trimethylimidazoliummethyl sulphate and 1-ethyl-3-methylimidazolium tetrafluoroborate (Sigma-Aldrich, St. Louis, MO, USA) were used. All the reagents used were of analytical grade.
Sodium cholate, sodium deoxycholate, sodium taurocholate, Tween 20, brij35, Tween 80, Triton X-100 and formaldehyde purchased from CDH (New Delhi, India) were used. All the surfactants were used as received.
Solution of biosurfactants was prepared in double-distilled water (DDW) to give a concentration of 5% (w/v).
S1 - Silica gel G
S2-S5 - Silica gel G impregnated with 0.1, 1.0, 5.0 and 10.0% 1-methylimidazolium chloride (aq), respectively.
S6 - Silica gel G impregnated with 5.0% 1,2,3-trimethylimidazoliummethyl sulphate (aq)
S7 - Silica gel G impregnated with 5.0% 1-ethyl-3-methylimidazolium tetrafluoroborate (aq)
M1 - 5.0% 1-Methylimidazolium chloride (aq)
M2 - Pure 2-methyltetrahydrofuran (purity, 99.0%)
M3 - Pure acetone (purity, 99.0%)
M4 - Pure dimethyl sulfoxide (DMSO; purity, 99.0%)
All the solvents were used as received.
For detection of anionic surfactants, 100-mg pinacryptol yellow (Fluka, Sigma-Aldrich, Steinheim, Germany) was dissolved in 100-mL hot water and sprayed on TLC plates to visualise yellow to orange fluorescence spots under long wavelength UV light (366 nm).
Preparation of 1-methylimidazolium chloride impregnated TLC plates
In pre-impregnation methods, plates were prepared by mixing silica gel G and aqueous solution of 1-methylimidazolium chloride of desired concentration (0.1~10.0%) in 3:1 ratio. The resultant slurry was mechanically shaken for 15 min and then coated onto glass plates with the help of a TLC applicator to give a layer of 0.25-mm thickness. The plates were first air dried at room temperature and then activated by heating at 100 °C for 1 h. After activation, the plates were kept in an air-tight chamber until used.
In post-impregnation method, activated silica gel plates were impregnated with desired concentration of aqueous solution of 1-methylimidazolium chloride (0.1~10.0%) by dipping silica gel plates in a solution of impregnate for a specific time period (20 min) followed by drying of plates at room temperature (25°C) and activation by heating for 1 h at 100°C. After activation, the plates were kept in an air-tight chamber until used.
In the present study, the TLC plates obtained by pre-impregnated method found better from a chromatographic point of view in terms of yielding more compact and well-resolved spots for biosurfactants. For example, the higher value (3.2 cm) of separated distance between the resolved spots of SDC and SC from their mixture on pre-impregnated TLC plates compared to the value of 2.1 cm on post-impregnated TLC plates is evident for better chromatographic performance of pre-impregnated method. Therefore, the detailed study was undertaken using pre-impregnating TLC plates.
The biosurfactant solutions (0.1 μl) were spotted on non-impregnated and impregnated TLC plates with micropipette at approximately 2 cm above the lower edge of the non-impregnated and impregnated TLC plates. The spots were dried at room temperature (25 ± 2°C). The glass jars containing the mobile phase were covered with lids and left for 10 min for saturation before introducing the plates for development. The plates were developed with listed solvent systems (M1-M4) to a distance of 10 cm from the origin in all cases. After development, the plates were detected by a spraying solution of pinacryptol yellow and viewing the location of biosurfactants under UV light (366 nm).
For the separation of biosurfactant mixtures, equal volume of each biosurfactant was mixed and 0.1 μL of the resultant mixture was applied onto 5%(aq) 1-methylimidazolium chloride impregnated TLC plates (S4). The plates were developed with M2, the spots were detected and R F values of the separated spots of the biosurfactants were calculated.
To understand the separation behaviour of biosurfactants in different impregnating liquids, 1-methylimidazolium chloride in S4 was replaced with 1,2,3-trimethylimidazolium methyl sulphate and 1-ethyl-3-methylimidazolium tetrafluoroborate. The resultant stationary phase systems (S6 and S7) were used for the chromatography of biosurfactants. R F values obtained with the use of these stationary phases were compared with those obtained with S4.
Surface structure and chemical composition of pure silica gel and silica gel impregnated with 5%(aq) 1-methylimidazolium chloride were studied with scanning electron microscopy (SEM) and energy-dispersive X-ray spectrophotometer (EDX).
In order to examine the mobility pattern of biosurfactants on S4, acetone (M3) and dimethyl sulfoxide (M4) were also used as eluents.
For investigating the interference of the presence of metal cations, metal anions, vitamins amino acids and non-ionic surfactants as impurities on the resolution of mixture, 0.1 μL of standard test mixture of biosurfactant solutions was spotted on the impregnated TLC plate followed by spotting of 0.1 μL of the metal cations, metal anions, vitamins and amino acids and non-ionic surfactants being considered as impurities. The plates were developed with M2 and detected, and R F values of the separated biosurfactants were calculated.
The limit of detection of separated biosurfactants were determined by spotting 0.1 μL of SC, SDC and STC of different concentrations on the impregnated TLC plates which were developed with selected mobile phase M2, and the spots were visualised using pinacryptol yellow reagent under UV light. This process was repeated with successive reduction in concentration of biosurfactant until no detection of biosurfactant was possible. The amount of biosurfactant just detectable was taken as the detection limit.
Results and discussion
Mobility of biosurfactants in terms of R F values on different stationary phases
R F value
Effect of concentration of 1-methylimidazolium chloride on Δ R F and separation factor (α) of biosurfactants
Concentration of 1-methylimidazolium chloride
Δ R F
Δ R F
0.1% 1-Methylimidazolium chloride
1% 1-Methylimidazolium chloride
5% 1-Methylimidazolium chloride
10% 1-Methylimidazolium chloride
Effect of substitution of 1-methylimidazolium chloride with different ionic liquids on R F of separated biosurfactants
Different ionic liquids
R F value
1, 2, 3-Trimethylimidazoliummethyl sulphate
Effect of substitution of 1-methylimidazolium chloride on Δ R F and separation factor (α)
Different ionic liquids
Δ R F
Δ R F
1, 2, 3-Trimethylimidazoliummethyl sulphate
Mobility of biosurfactants in terms of R F value on S 4 with different mobile phases
R F value on S 4
Effect of interference on Δ R F , separation (α) and resolution (R S ) factors of the separated biosurfactants
Δ R F (0.52)
R S (48.0)
Δ R F (0.36)
R S (57.7)
Triton X 100
Unimpregnated silica gel TLC plates (S1)
On pure silica static flat phase (S1) using 5%(aq) 1-methylimidazolium chloride (M1) as mobile phase, SDC remains at the point of application (R F~0.02) whereas SC and STC were not detected.
SC (R F = 0.54) and SDC (R F = 0.65) exhibit high mobility and STC remains at the point of application (R F~0.02) with 2-methyltetrahydrofuran (M2) as mobile phase and S1 as stationary phase. Binary separations of these biosurfactants were achieved with the use of S1 and M2 as stationary and mobile phases (Table 1).
Impregnated silica gel TLC plates (S2-S5)
SC and SDC showed differential migration (R F = 0.48, 0.76) while STC remains at the point of application (R F~0.03) on using S2 (silica gel impregnated with 0.1%(aq) 1-methylimidazolium IL) as stationary phase and M2 as mobile phase. Though the mutual separation of these SC, SDC and STC was possible with this TLC system, but the spots lack clarity and compactness; hence, concentration of impregnant was increased up to 10%.
At 1.0% concentration of impregnant (S3), the mutual separation of three surfactants SC (R F = 0.53), SDC (0.83) and STC (R F~0.02) was also possible with slightly improved compactness of spots.
The better resolved spots of surfactants (SC, SDC and STC; R F = 0.48, 0.89 and 0.03) from their three-component mixture were realised with M2 mobile phase and silica gel G impregnated with 5%(aq) 1-methylimidazolium chloride (S4) stationary phase. Therefore, this TLC system was considered the best system for the resolution of coexisting SC, SDC and STC biosurfactants.
The higher concentration of impregnant exceeding to 5.0% was not found suitable because SC and SDC could not be detected on the use of silica gel impregnated with 10%(aq) 1-methylimidazolium chloride (S5) as stationary phase in combination with M2 mobile phase (Table 2).
Thus, the TLC system (S4-M2) was most favourable for the resolution of coexisting SC, SDC and STC biosurfactants (Figure 1).
Silica gel impregnated with other ionic liquids (S6-S7)
Compared to S4 (R F = 0.79) (Tables 3), the lowering in R F value of SDC on S6 (R F = 0.53) results in the inferior resolution of SDC from SC.
SEM and EDX
Mobility of biosurfactants with other aprotic polar solvents (acetone and DMSO)
Effect of replacement of silica with other sorbents
Effect of interference
Limit of detection
Composition of Fluarix influenza vaccine
It can be conclude that the use of 1-methylimidazolium chloride in stationary phase as impregnant and 2-methyltetrahydrofuran as mobile phase provides favourable environment for better separation possibilities of biosurfactants. The chosen TLC system was successfully applicable for the identification of sodium deoxycholate in a formulated sample containing the components of influenza vaccine. Another important aspect of this study is the use of green solvent as impregnant in stationary phase in addition to the use of green mobile phase making the proposed TLC system environment friendly.
volatile organic compounds
scanning electron microscopy
energy-dispersive X-ray spectrophotometer
The authors are thankful to the Department of Applied Chemistry and the University Grants Commission, New Delhi, for providing research and financial assistance respectively to both authors who are working as UGC-BSR Faculty Fellow and research scholar.
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