Pang Xiuyan, Sun Hanwen, Feng Yongqiang, Liu Xiulan, Shen Shigang
(Key laboratory of analytical science and technology of Hebei province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002,China)

Abstract The retention behaviors of aromatic sulfonates are discussed based on the reversed-phase ion-pair chromatography. The response of relative retention factor to the concentration of ammonium tetrabutyl bromide, tetramethylene-oxide permitted to better fit the data and to build reliable predictive models respectively. The relationship between the content of tetramethylene-oxide and the polarity， the surface tension of the mobile phase has been investigated with fluorescence probe and maximum bubble method, and the results testify that tetramethylene-oxide can obviously decreasing the surface tension and polarity. Thus it results the reduction of the sulfonates' retention in the chromatographic system. The influence of pH of the mobile phase on the retention was also discussed.
Keywords The reversed ion-pair chromatography, sulfonates, tetramethylene-oxide, retention behavior, polarity, surface tension

1 INTRODUCTION
Aromatic sulfonates such as benzene sulfonates and naphthalenesulfonates are widely used as intermediates. The excellent hydrophilic properties and low biodegradability makes them potentially hazardous ^[1]. Thus the detection of sulfonates becomes popular ^[2,3]. Many analytical techniques have been proposed for determining these compounds in water. Gas chromatography analysis needs a previous step to convert them into volatile derivates ^[4]. When capillary electrophoresis is used, it gives better result particularly using laser induced fluorescence detection ^[5]. The preferred technique, however, is still the reversed phase liquid chromatography (RP-LC) combined with UV ^[6], fluorescence^[7] or MS^[8,9]，for it can offer a higher separation efficiency than that by capillary electrophoresis^[3].
    In the RP-LC analysis, the main factors, which influence the retention of analytes, are pH of the mobile phase and concentration of organic modifiers. Nikitas et al. studied the retention mechanism of the solute molecules from aqueous with the addition of organic modifier to the mobile phase ^[10-12], and the combined effect of pH and organic modifier concentration on the retention were described with six equations ^[12]_. An attractive analysis method of ionic samples is the technique commonly referred to as ion-pair liquid chromatography (IPLC). In the RP-IPLC, pH of the eluant is adjusted in order to encourage ionization ^[13], and the eluent pH is recommended to be above the analytes' pK_a value, in order to make sure dissociation of the acidic groups and strong ion-pair formation. Meanwhile an increasing counter-ion concentration further intensifies the retention behavior ^[14]. Electrolyte concentration (inorganic modifier) further influences the retention, and it decreases with the increasing electrolyte concentration ^[15].
    In the RP-IPLC analysis of aromatic sulfonates, Leon et al. reported the detection of  the C₄ - C₁₃ benzene-sulfonic acids ^[16], though gradient solvents were used, the retention time of C₁₃ benzene-sulfonic acid was still near 50min. Under the condition of 10%-50% methanol-water gradient solvent, 7mM TBA and pH 6.5, Gimeno et al. had detected 14 aromatic sulfonates^[17]. The retention time of 4-aminobenzenesulfonate， benzenesulfonate and 3-nitro-benzenesulfonate were 10min, 38min and 52min respectively. It is necessary to establish fast separation technique to improve the detection sensitivity and accuracy especially in the quantitative and qualitative analysis according to the peak height, area and retention time. Robert et al. had replaceed the 250 mm long column with a "fast and short" column (75×4 mm I.D., 4mm particle diameter), with the retention is reduced in half ^[6].
    The purpose of this paper is， based upon the former works ^[18,19], to give further study on the retention behavior of aromatic sulfonates with THF together with methanol as organic modifier, and search for the influence of tetramethylene-oxide THF on polarity and surface tension of the mobile phase, in order to provide reliable theoretical bases for the rapid detection of sulfonates.
    In this research, the polarity and surface tension of the mobile phase has been investigated with fluorescence probe and maximum bubble method ^{[20, 21]}, respectively. The research provides important evidence for the relationship of the retention behavior and THF concentration.

2. EXPERIMENTAL
2.1 Reagents
Ammonium tetrabutyl bromide(analytical grade), tetramethylene-oxide (analytical grade, fresh distillated before use), methanol (HPLC quality). The following sulfonated compounds were analytical reagent and purchased from Beijing: 4-aminobenzenesulfonate, benzenesulfonate, 2-naphthol-6, 8-disulfonate, 2- naphthol-3, 6- disulfonate, 3-nitro-benzenesulfonate, dibenzal-4-sulfonate, methyl orange. All the other chemicals were commercial analytical reagent. Redistilled water was used for preparing the mobile phase.
    For the experiments, a stored water solution of 1g· L^-1 of each compound was prepared. Standard solutions of each compound in the concentration of 0.001-0.1 g· L^-1 were prepared with the mobile phase as diluent, respectively. The pH value of the mobile phase was adjusted with K₂HPO₄ or KH₂PO₄.
2.2 Instrument
The chromatographic condition was the same as that mentioned in the former work ^[19]. The pH of the mobile phase was detected with PHS-3C acidometer. The fluorescence intensity of chrysene was detected under the condition of l_ex/l_em= 365/428nm in the solution of water-methonal-THF with RF- 540 (Shimadzu, Japan), and the surface tension of the mobile phase was mensurated with simple experimental equipment.
    The retention behavior of sulfonates in 15:85 methanol-water (v/v), and 30:70 methanol-water (v/v) mobile phases containing different content of THF was detected, respectively.
    The influence of THF content on polarity and surface tension of the mobile phase was studied.

3 RESULTS AND DISSCUSSION
3.1 The influence of THF on the retention of sulfonates                               
THF could more efficiently ameliorate retention time of components than methanol ^{[18, 22]}, meanwhile, it possessed low viscosity and weak UV absorption under the detected wavelength. Figure 1 described the relationship between the sulfonates' relative retention factor r and THF content C in methanol-water mobile phase. The data was managed with non-linear imitation method, and models of Ln r =A - B·C_THF were obtained when the concentration of THF was low (Table 1). The linear relationship between C_THF and Ln r indicated that r decreased at the index rate with the increasing of C_THF. The models were similar to that of methanol and acetonitrile on sulfonic acid ^[23]. The concentration of THF, which could give r a 50% reduction, was listed in Table 2. All this indicated the remarkable amelioration of retention time of analytes.

                                                  a

                                                   b

Figure 1 The influence of THF concentration on sulfonates' relative retention factor
a  …the 15:85 methanol - water eluent containing different percent of THF,   b  …the 30:70 methanol- water eluent containing different percent of THF
(1) 4-aminobenzenesulfonate (2) benzenesulfonate   (3) 2-naphthol -6,8-disulfonate   (4) 2- naphthol-3,6- disulfonate (5) 3-nitro-benzenesulfonate (6) dibenzal-4-sulfonate (7) methyl orange

Table 1 The retention models of sulfonates under the influence of THF

* The models in the second list corresponding to the mobile phase of 15:85 methanol-water, and the third corresponding to the mobile phase of 30:70 methanol- water
r …the relative retention factor of sulfonates, C …the volume percent of THF in the mobile phase.

Table 2 The content of THF causing relative retention factor r a fall of 50%*

* The second list corresponding to the mobile phase of 15:85 methanol-water, and the third corresponding to the mobile phase of 30:70 methanol- water

    Organic modifier could shorten retention time and ameliorate separation selectivity through reducing the polarity of the mobile phase and abating the column capacity ^[24]. To testify the influence of THF on the polarity of the eluent, the fluorescence probe method with chrysene as fluorescence reagent was employed. The fluorescence intensity of chrysene was detected under the condition of l_ex/l_em= 365/428nm with water, acetonitrile, methanol, THF, acetone，hexane as solvent, respectively. The results of 42.1, 34.8, 34.6, 25.4, 26.8 and 17.5 illustrated that the emission strength of chrysene increased with the polarity of the solvents. When the solvent, which had an increasing volume ratio of THF to methanol-water, was used, a gradually depressed intensity was gained (showed in Figure 2.) The results indicated that THF could efficiently reduce the polarity of the eluent, which resulted the apparent reduction of sulfonates' retention time.

Figure 2 The fluorescence intensity of chrysene in different volume ratio of THF -methanol-water solution
1…15:85 methanol-water， 2…30:70 methanol-water
C（%）…the volume percent of THF in the mobile phase.

    According to Bidlingmeyer ^[25], there was higher surface tension between stationary phase and mobile phase. The solvent, which could modify surface tension, could also adjust the retention behavior of analytes. To study the influence of THF, the surface tension of methanol-water-THF solution was detected with the maximum bubble method. The detected solution had an increasing volume ratio of THF to methanol-water. The results were showed in Figure 3. In 15:85 methanol-water solution, the addition of 20% THF made a 16% reduction in the surface tension. While in 30:70 methanol-water solution, an 18% reduction was gained. The results declared that THF could evidently ameliorate the surface tension, and it could adjust the retention behavior of the components.

Figure 3 The influence of THF on surface tension
（1）^... 15:85 methanol - water， （2）^...30:70 methanol - water
C（%）^... the volume percent of THF in the mobile phase.

3.2 The influence of TBA on retention behavior
In the analysis of ion-samples, the addition of counter-ion was necessary. Wang studied the influence of TBA on six organic acids^[14], but the reaction character of sulfonates with TBA as ion-pair reagent had not been studied systemically. In the experiment, the effect of TBA content on the retention of the seven analytes was showed in Figure 4. With the increasing of TBA concentration, a shortened retention time was gained. When the TBA content was lower than the maximum column capacity, there was a linear relationship between the Ln r and LnC_TBA. The relation was described in Table 3, and Zou reported the similar results in the studying of sulfonic acid ^[26]. But different analyte had different effectual column capacity, 3-nitro-benzenesulfonate, dibenzal-4-sulfonate and methyl orange had lower value than the others.
3.3 Influence of pH on retention feature
When the ion-sample was separated by RP-IPLC, pH of the eluant is adjusted in order to encourage ionization ^{[13, 14]}. The effect of pH value on sulfonates' retention was determined and showed as Figure 5. The accrescent pH value caused sulfonates' a minished retention time. The effect of pH value on benzenesulfonate was very slight, while the change of orange was more notable.
3.3 The separation of aromatic sulfonates
In the separation of sulfonates, the following chromatographic conditions were employed: methanol - water (30:70, v/v)-THF (5%)-TBA (3 mmol/L), pH 6.5 with an isocratic flow rate of 1.0 mLmin^-1. The chromatograms were recorded as Figure 6, and the retention time of 4-aminobenzenesulfonate， benzenesulfonate， 2-naphthol-6，8-disulfonate，2- naphthol-3，6- disulfonate and 3-nitro-benzenesulfonate was 3.14min, 4.33min, 4.99min, 5.30min and 7.45min, respectively. But the retention time of dibenzal-4-sulfonate and methyl orange exceeded 40min. When the mobile phase was changed into methanol-water (30:70, v/v)-THF (16%)-TBA (3 mmol/L), the increasing of THF content greatly shorten the retention of dibenzal-4-sulfonate and methyl orange, a retention time of 6.04min and 9.17min was detected respectively (Figure 7).

Figure 4. The influence of TBA concentration on sulfonates' retention behavior
(The mobile phase was methanol- water (30:70, v/v)- pH 6.5 and 5%THF for compound 1-5, 16% THF for compound 6-7)
(1) 4-aminobenzenesulfonate (2) benzenesulfonate (3) 2-naphthol-6,8-disulfonate (4) 2- naphthol-3,6- disulfonate (5) 3-nitro-benzenesulfonate (6) dibenzal-4-sulfonate (7) methyl orange

Table 3  The relationship between relative retention factor and TBA Concentration