Determination of aromatic sulfonated compounds by reversed-phase ion-pair chromatograohy with tetramethylene-oxide as organic modifier

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

Received Jan. 15, 2004; Supported by the Natural Science Foundation of Hebei province, China.(No.203110)

AbstractA reversed-phase ion-pair chromatography method with ammonium tetrabutyl bromide as ion-pairing reagent and UV detection has been developed for aromatic sulfonated compounds. It was testified that the concentration of ion pair reagent, pH and the volume ratio of tetramethylene-oxide in mobile phase played important roles in the separation. Linearity regression equations along with linearity ranges, correlation coefficients of 0.9991-0.9996 and detection limit for determination of seven sulfonated compounds were offered. To achieve the detection of analytes in low concentration water samples, the ion-pair extraction technique was employed with ammonium tetrabutyl bromide (TBA) as ion-pair reagent and dichloromethane as extractant. Recoveries of the method were between 96.8 and 101.7%, and relative standard deviation was below 7.6%. Different concentration of 3-nitro-benzenesulfonate, dibenzal-4-sulfonate and methyl orange were detected from rainwater, tap water and electrodialysised water sample, respectively.

Aromatic sulfonates such as benzene sulfonates and naphthalenesulfonates are widely used as intermediates in the chemical industry, particularly in the production of pharmaceuticals, dyes, tanning agents, pesticides, cement, concrete and so on. Due to their excellent water-solubility and hydrophilic properties, it is difficult to remove them completely from waste water in the water treatment works and they are discharged into the aquatic environment[1]. The toxicology and environmental ecology research is just underway[2,3]. Low biodegradability makes them potentially hazardous[4]. Various analytical techniques have been proposed for determining these compounds in water. Gas chromatography analysis needs a previous step to convert them into volatile derivates[5]. When capillary electrophoresis is used, good results have been obtained particularly using laser induced fluorescence detection[6]. The preferred technique, however, is still ion-pair liquid chromatography with UV [7], fluorescence[8] or MS[9]
The detection limits obtained with these techniques are not low enough for them to be determined in real water samples. For this reason, an enrichment step is necessary. Ion-pair extract technique is a method to improve the solubility of ionic compounds in organic solvent through the formation of neutral associated compounds with ion-pair reagent[10]. To improve the detection sensitivity and veracity of sulfonates, it is necessary to establish the fast analysis method[11]. The aim of this paper is to develop a method for determining a mixture of benzene sulfonates, and naphthalenesulfonates. The method is based on ion-pair liquid chromatography with UV detection, coupled with ion-pair extraction. To our knowledge, the simultaneous detection of mixed benzene and naphthalene sulfonate compounds (4-aminobenzenesulfonate,benzenesulfonate,2-naphthol-6,8-disulfonate,2- naphthol-3,6- disulfonate,3-nitro-benzenesulfonate, dibenzal-4-sulfonate, methyl orange) in water samples has scarcely been studied.

2.1 Instrument

The sensitive UV absorption wavelengths were taken by a Tianmei 8500 spectrophotometer (Shanghai, China). The pH were detected with a Tianmei PHS-3C acidometer (Shanghai, China).
The chromatographic system consisted of the Shimadzu corporation commercial components: a solvent-delivery pump, a SPD-6AV UV-VIS detector, operating at 220 nm. The data were collected and analyzed by using CR-3A system (Kyoto, Japan). Separations were performed on a C18 column (25.0 cm×0.46 cm 5m )from Elite( Elite Scientific Instruments Co. Ltd, Dalian, China) at ambient temperature(about 19C) and a flow rate of 1.0mLmin-1. Sample volume of 5L was injected in the “push loop”mode.
2.2 Reagents and the Preparation of Samples
Ammonium tetrabutyl bromide(analytical grade), tetramethylene-oxide (analytical grade, fresh distillated before use), methanol (HPLC quality), and 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. Double-distilled water was used for preparing the mobile phase.
For the experiments, a stored water solution of 1g L-1 of each compound is prepared. Standard solutions of each compound in the concentration of 0.001-0.1g L-1 are prepared with the mobile phase as diluent,respectively.
Samples were collected with 500 mL pre-cleaned amber glass bottles, filtered through a 0.45m membrane filter and kept at 4until analysis. A 20 mL of the sample was enriched in a plugged-glass tube through the addition of a definite mass of extractant and ion-pair reagent under a definite pH value, and the extracts were analyzed with the mentioned chromatographic system.

3.1 The selection of ion pair reagent and its concentration

High performance liquid chromatography was not suited for either efficient retention or separation of the sulfonates. The addition of ion-pairing agents at appropriate concentration made them have efficient retention. Ammonium tetrabutyl bromide (TBA) had relatively low molecule mass than other ion-pair reagent, which was more beneficial to improve the separation selectivity of components with similar structure. The effect of TBA concentration on the retention of seven analytes was examined. The results were showed in Figure 1 and it illustrated that the sulfonated compound had a shortened retention time with the increasing of TBA content, and different component had different efficient column capacity.


Fig.1 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-sulfonate7)methyl orange

3.2 Influence of pH on retention behavior
To insure the formation of ion-pair, the pH value of mobile phase was adjusted ranging from 3.5 to 7.5 by adding phosphoric acid or sodium hydrogen phosphate[12]. The raising pH value resulted in the reduction of retention time as showed in Figure 2. That might be caused by the increasing negative charge of phosphate, which promoted the complexing of phosphate with TBA, then reduced the complexing of TBA with sulfonate negative ion[13]. The affection of pH value on benzenesulfonate was very slight, but the change of methyl orange was more notable. Through the adjustment of pH value, good separation of sulfonates could be obtained.


Fig. 2 The influence of pH value on sulfonates’ relative retention factor
(The mobile phase was methanol- water (30:70, v/v)- TBA (3mmol/L) and 5 % THF for compound 1-5, 16 % THF for compound 6-7)
(1) 4-aminobenzenesulfonate () benzenesulfonate () 2-naphthol-68-disulfonate (4) 2- naphthol-36- disulfonate () 3-nitro-benzenesulfonate () dibenzal-4-sulfonate () methyl orange
Fig. 3 The influence of THF concentration on sulfonates’ relative retention factor
((The mobile phase was methanol- water (30:70, v/v)- TBA (3mmol/L), pH 6.5)
(1) 4-aminobenzenesulfonate(2) benzenesulfonate(3) 2-naphthol -68-disulfonate(4) 2- naphthol-36- disulfonate(5) 3-nitro-benzenesulfonate  (6) dibenzal-4-sulfonate(7) methyl orange

3.3 The influence of organic reagents on retention
Aromatic sulfonates all possessed the hydrophilic group and lipophilic group, which demanded the eluent should contain both water and organic solvent. When the methanol content was 30%, the retention time of 3-nitro-benzenesulfonate was about 30min, and dibenzal-4-sulfonate, methyl orange was larger than 1h, respectively. With the increasing of methanol concentration, the retention times were shortened, but the separation of sulfonates was destroyed. THF was a more efficient solvent than methanol in reducing the retention of components[14], and it possessed low viscosity and UV absorption wavelength. In the experiment, the retention behavior of sulfonates in 30:70 methanol-water (v/v) mobile phase containing different content of THF was studied. According to the experimental results (showed in Figure 3) we knew that THF could effectivelyreduce the retention time of sulfonates. This was caused by the reduction of polarity of the mobile phase and abasing the column capacity.
3.4The Separation of Sulfonates
In the separation of sulfonates, the concentration of THF, methanol, TBA and the pH value of the mobile phase was important influencing factor, and the content of THF was the crucial factor in speedy separation. The mobile phase was methanol- water (30:70, v/v)-THF (5%)-TBA (3 mmol/L), pH 6.5. Under isocratic flow rate of 1.0 mLmin-1, the chromatograms were recorded as Figure 4, and the retention time of 4-aminobenzenesulfonate, benzenesulfonate, 2-naphthol-68-disulfonate2- naphthol-36- disulfonate and 3-nitro-benzenesulfonate was 3.14min, 4.33min, 4.99min, 5.30min and 7.45min, respectively. The analysis was much faster than that of Gimeno[15], who used a mobile phase of methanol and water under different gradients, with the retention time of 4-aminobenzenesulfonate, benzenesulfonate and 3-nitro- benzen esulfonate was 10min, 38min and 52min, respectively. When the mobile phase was changed into methanol-water(30:70, v/v)-THF(16%)-TBA (3 mmol/L), the retention time of dibenzal-4-sulfonate and methyl orange was 6.04min and 9.17min (Figure 5), respectively.
FigThe chromatograms of () 4-aminobenzenesulfonate   () benzenesulfonate () 2-naphthol-68-disulfonate () 2- naphthol-36- disulfonate and () 3-nitro-benzenesulfonate
(The mobile phase was methanol- water (30:70, v/v)-THF (5%)-TBA (3 mmol/L), pH 6.5.)
R,1= 3.14min, tR,2= 4.33min, tR,3= 4.99min, tR,4= 5.30min, tR,5= 7.45min
Fig. 5 The chromatograms of () dibenzal-4-sulfonate and () methyl orange
(The mobile phase was methanol- water (30:70, v/v)-THF (16%)-TBA (3 mmol/L), pH 6.5.)

3.5 Linearity and Detection Limit
The relation between peak area and mass of the component was examined. The linearity regression equations along with linearity ranges, correlation coefficients and detection limit for determination of the seven components were given in Table 1.

Table 1 The linearity and detection limit

Components Regression equation Linearity (g) Correlation coefficient Detection limit(ng)
4-aminobenzenesulfonate A=-372.4+50536.7X 0.03-6.0 0.9992 3.9
benzenesulfonate A=751.6+14078.9X 0.015-2.5 0.9994 8. 8
2-naphthol-68-disulfonate A=-236.7+43146.7X 0.035-8.0 0.9991 4.1
2-naphthol-36-disulfonate A=514.6+30292.4X 0.03-5.0 0.9995 3.3
3-nitro-benzenesulfonate A=1380.8+17128.5X 0.025-8.0 0.9996 3.2
dibenzal-4-sulfonate A=554.6+8339.7X 0.03-6.0 0.9994 2.3
methyl orange A=-249.3+71253.9X 0.02-4.0 0.9996 3.9

A:the peak area, X: the mass of the components(g).

3.6 The enrichment of samples
To achieve the determination of sulfonates existed in water samples, ion-pair extraction was carried out with TBA as ion-pair reagent and dichloromethane as extractant. The enriching condition was optimized by determination the influence of TBA concentration, volume ratio of extractant to sample and pH value. The effect of TBA on extraction recovery was more important than pH. When TBA equaled to 1.4 mg mL-1, the maximum recovery of single extraction was obtained (showed in Figure 6). Table 2 showed the results under the optimum condition of TBA (1.4 mg mL), dichloromethane-sample (1:20,v/v) and pH 6.0-7.0. Recoveries of the method were between 96.8 and 101.7% (the data listed in the Table 2. had been corrected with compound’s extraction recovery), and relative standard deviation was below 7.6%.
Fig. 6 The influence of TBA concentration on the extraction recovery
(The extraction was carried out under the condition of dichloromethane-sample (1:20,v/v) and pH 6.5)
(1) 4-aminobenzenesulfonate(2) benzenesulfonate(3) -2-naphthol-68-disulfonate(4) 2- naphthol-36- disulfonate(5) 3-nitro-benzenesulfonate  (6) dibenzal-4-sulfonate(7) methyl orange

Table 2 Recoveries of the component

Components 4-amino benzene sulfonate benzene sulfonate 2-naphthol-68- disulfonate 2-naphthol-36-disulfonate 3-nitro-
dibenzal-4-sulfonate methyl orange
Recoveries (%) 98.5 98.8 98.2 97.6 101.7 96.8 99.8
RSD% 4.96 4.96 3.4 6.9 2.5 5.7 7.6
image image
Fig. 7 3-Nitro-benzene-sulfonate detected from rain water
(The mobile phase was the same as that of Fig. 4)
Fig. 8 Dibenzal-4-sulfonate and methyl orange detected from rain water
(The mobile phase was the same as that of Fig. 5)

The method was applied to the analysis of rainwater, tap water and electrodialyzed water, respectively. The content of sulfonates in samples was detected byextrapolation with the results listed in Table 3. 3-Nitro-benzenesulfonate, dibenzal-4-sulfonate and methyl orange were detected as given in Figure 7 and Figure 8.

Table 3 The detected sulfonates in samples

Sample The content of the components(g/L)
3-nitro-benzene-sulfonate dibenzal-4-sulfonate methyl orange
Rainwater 23 71 30
Tap water 68 18 35
Electrodialyzed water 13 7.6 2.4

In the fast analysis of dissociable compounds based on the reversed-phase ion-pair chromatography, THF was an effective organic modifier for shortening analyzing time. Ion-pair extraction of water sample made it possible for the detection of low concentration sulfonates. The proposed method was simple and easy with good accuracy and precision. It provided an effective detection measurement for the environmental evaluation. If the analysis was performed by way of gradient elution, the simultaneous and fast analysis of the seven compounds can be performed.