Simultaneous Determination of Nonylphenol and Nonylphenol Ethoxylates in Wastewater Samples from Biodegradation Process by High Performance Liquid Chromatography Method

The degradation products of nonylphenol ethoxylates (NPnEO), nonylphenol (NP) and shortchain NPnEO are representative of environmental endocrine disruptors. They possess strong lipophilicity, toxicity, cumulative property and estrogenic effect. They can pollute the environment, cause body precocious, and induce the body’s estrogen-sensitive cancer cell proliferation. A fast method using high performance liquid chromatography (HPLC) was developed to simultaneously quantify NP and 11 kinds of NPnEO in wastewater samples. The influence of mobile phase composition, mobile phase ratio, mobile phase flow rate, column temperature and sample injection volume on the separation effect was studied. Under the optimized conditions, NP and 11 kinds of NPnEO were separated successfully within 35 min. The method showed good linearity for NP and 11 kinds of NPnEO (n = 1-11). The linear correlation coefficients for the standard curves were 0.97200.9999. The precision degree of the method was reliable and all the relative standard deviation (RSD) values (n = 0-11) obtained were less than 5.0%.


Introduction
In recent years, it was discovered that the endocrine disruptors in the environment could interact with the endocrine system in organism even with a small dosage.Endocrine disruptors perturb the synthesis, secretion, transport, metabolism, binding action, or elimination of endogenous hormones. 1 These endocrine disruptors not only pollute the environment but also cause a serious threat to people's health.][3][4][5] Nonylphenol (NP) and nonylphenol ethoxylates (NPnEO) of short chain (n = 1, 2) are typical endocrine disruptors. 2,6NP and short chain nonylphenol ethoxylates (SC-NPnEO) possess strong lipophilicity, toxicity, cumulative property and estrogenic effect. 2,6,7][9] Currently, the harmless treatment of NPnEO has become a hot topic in the environmental field.][12] The anoxic-oxic activated sludge process (AOASP) is mainly combined with an anoxic unit, an oxic unit and a settlement unit. 11The wastewater is introduced into the anoxic unit firstly.Some operations such as stirring are performed in anoxic unit to keep the activated sludge suspending.Then the wastewater flows into the oxic unit.Aeration is conducted in oxic unit to supply oxygen for the activated sludge bacteria.The circulation is carried out between the anoxic and the oxic unit to strengthen the treating effect such as NPnEO degradation.The settlement unit is used for the separation of treated water and activated Vol. 28, No. 12, 2017 sludge.A lab-scale AOASP was established to treat the synthetic long chain mixed NPnEO wastewater.In order to detect and quantify the composition of NP and NPnEO in wastewater samples from the AOASP, a method aimed at the simultaneous determination of NP and NPnEO (n = 1-11) was established in this study.
5][16] In this study, according to the properties of target detection objects, the economical and practical HPLC method was chosen to determine NP and NPnEO (n = 1-11) in wastewater samples from biodegradation process.The influence of mobile phase composition, mobile phase ratio, mobile phase flow rate, column temperature and sample injection volume on the separation effect was investigated in detail.The linearity, linear correlation coefficient, relative standard deviation (RSD) and average recovery of this method were also studied.A simple, fast and reliable measuring method for NP and NPnEO (n = 1-11) was established and it is expected to offer helps for related researches.
Isopropanol (C 3 H 8 O), n-hexane (C 6 H 14 ) and dichloromethane (CH 2 Cl 2 ) were chromatographically pure chemicals from Tianjin Concord, China.Standard reagents of NP and mixed NPnEO (average n ca. 2 and 5) were from Tokyo Chemical Industry Co. LTD, Japan.The standard stock was prepared using isopropanol with NP, mixed P2EO and mixed NP5EO concentrations of 1074, 2140 and 4920 μg mL -1 , respectively.During the optimization process, the used mixed standard solution was composed of: 10 times diluent of NP stock, 5 times diluent of NP2EO stock and 2.5 times diluent of NP5EO stock.Finally the NP, mixed NP2EO and mixed NP5EO concentrations were 107.4,428.0 and 1968 μg mL -1 , respectively.

Pretreatment of wastewater samples
The used wastewater samples were the synthetic long chain mixed NPnEO influent and the effluent of the lab-scale AOASP.The wastewater samples were centrifuged for 5 min under 6000 rpm to remove the suspended solids.Then the supernatant was filtrated by a 0.45 μm organic membrane.50 mL of diluted filtrate (influent for 12.5 times, effluent without dilution) was pipetted into 125 mL separating funnel, then 0.5 mL of 1 mol L -1 HCl and 2.5 g NaCl were added, and shaken well.After that, it was added 5 mL of dichloromethane, shaken for 2 min, stood for 20 min, and then the lower organic phase was collected, which was centrifuged at 5000 rpm for 10 min.Afterwards, the centrifuged upper water phase was moved back into the separating funnel, 5 mL of dichloromethane was added, and then the extraction and centrifugation processes were repeated.The centrifuged lower organic phase of two centrifugation processes was filtered to remove the floccules, then the filtrate was evaporated at 40 °C to dryness, the residue was dissolved with isopropanol, made to 2 mL, and filtered by 0.45 μm organic filtration membranes.The pretreated sample was finally achieved.The influent was concentrated for 2 times and the effluent was concentrated for 25 times.
In the Results and Discussion section (Wastewater sample determination sub-section), the recovery experiment is described.A certain concentration of standard solution was added into the influent.This solution was pretreated by the pretreatment method and then measured by the decided HPLC-UV method.The results proved the validation of the pretreatment method.

Chromatographic separation conditions
The mobile phase system, mobile phase ratio and gradient elution procedure, mobile phase flow rate, column temperature, injection volume were optimized in this study.The used HPLC-UV detection wavelength was 277 nm.
T h e t e s t e d m o b i l e p h a s e s y s t e m s w e r e n-hexane:isopropanol, n-hexane:dichloromethane and isopropanol (A):n-hexane (B):dichloromethane (C).The optimized process of mobile phase ratio and gradient elution procedure were operated step by step.The detailed process was shown in the Results and Discussion section (Selection of mobile phase ratio and gradient elution procedure sub-section).The tested flow rates were 0.8, 0.9, 1.0, 1.1 and 1.2 mL min -1 .The tested column temperatures were 23 (room temperature), 30, 32 and 34 °C.The tested injection volumes were 5, 10, 20 and 25 μL.

Selection of mobile phase system
The Hypersil APS-2 amino chromatographic column used in this study was a normal-phase chemical bonding chromatographic column.In normal-phase chromatography, the elution capacity of mobile phase system increases with the solvent polarity.The appropriate selection of mobile phase system can significantly improve the selectivity of the measured components.In order to obtain suitable solvent strength, a binary or ternary solvent system is normally used as mobile phase.The used solvent can be divided into the based primer and the eluent.In normal-phase chromatography, low polarity solvents such as n-hexane, benzene, and chloroform are usually adopted as the based primer and polar solvents, such as ethers, esters, alcohols and ketones, are commonly selected as the eluent.Two binary mobile phase systems (n-hexane:isopropanol, n-hexane:dichloromethane) and one ternary mobile phase system (isopropanol:n-hexane:dichloromethane) were investigated in this study.The separation results of mixed standard solution were shown in Figure 1.
Figure 1a showed that the separation between NP and NP2EO was not clear and was difficult to improve with the changing of mobile phase ratio.NP and adjacent NPnEOs still have not been separated by the n-hexane:dichloromethane system in Figure 1b.The long chain NPnEOs mixed together and was difficult to separate.With the isopropanol:n-hexane:dichloromethane system, NP and adjacent NPnEOs have been fully separated.Long chain NPnEOs also presented a clear separation and shorter retention time.With the further optimization of chromatographic conditions, it was possible to separate NP and NPnEOs clearly and rapidly.Therefore, the isopropanol:n-hexane:dichloromethane ternary system was chosen as the mobile phase system in this study.

Selection of mobile phase ratio and gradient elution procedure
It was found that the small variation of dichloromethane ratio in the isopropanol (A):n-hexane (B):dichloromethane (C) system would affect the retention time and the separation degrees between NP and adjacent NPnEOs.Three gradient elution procedures with different dichloromethane ratios (A:B:C linear changed within 30 min: from 1:96:3 to 10:87:3, from 1:95:4 to 10:86:4, from 1:94:5 to 10:85:5) were performed to investigate the separation of NP1EO, NP2EO, NP and NP3EO.Under the dichloromethane ratio of 4% (Figure 2), NP1EO, NP2EO and NP achieved complete separation, while NP and NP3EO showed a little overlap, which could be improved by changing the isopropanol ratio in the gradient elution procedure.The optimal dichloromethane ratio was chosen as 4% and the initial A:B:C used was 1:95:4.
In gradient elution process, the changing rate of strong eluent ratio affects the separation degree of different components.A better separation degree will be achieved under bigger changing rate.Four gradient elution procedures with different isopropanol ratios (initial A:B:C = 1:95:4, linear changed within 14 min under different changing rates of isopropanol ratio: 0.20, 0.25, 0.30, 0.35% min -1 , then linear changed to 15:81:4 within 1 min and maintained for 10 min) were conducted.The results showed that all the separation degrees between NP1EO, NP2EO, NP and NP3EO achieved 1.4 under the isopropanol ratio changing rate of 0.25% min -1 .The corresponding chromatogram is shown in Figure 3.
According to the separation degree between NP2EO and NP, the long retention time of long chain NPnEOs in Figure 3 and the maximum of n, the following procedures of the three gradient elution were carried out: initial A:B:C = 1:95:4, linear changed to 4.5:91.5:4within 14 min, in 14-15 min linear changed to 17:79:4, maintained for 7 min, in 22-36 min linear changed to 45:51:4, 59:37:4 or  The results showed that all the separation degrees of last two procedures were larger than 1.0 and achieved the minimum separation requirement.But the changing rate (to 59:37:4) in 22-36 min presented shorter total retention time.The final gradient elution procedure was obtained successfully.The corresponding chromatogram is shown in Figure 4 and all 12 components were separated within 35 min.

Selection of mobile phase flow rate
Based on the rate theory, the plate height is proportional to the mobile phase flow rate.Low mobile phase flow rate in HPLC can reduce the plate height and thus improve the column efficiency.However, the flow rate should not be too slow due to the broadening of chromatography peak and the increasing of retention time.Five mobile phase flow rates, 0.8, 0.9, 1.0, 1.1 and 1.2 mL min -1 , were used to investigate the influence on retention time and theoretical plate number.The results showed that the retention time of these components correspondingly reduced with the increasing of flow rate, but the separation degrees presented no significant differences.The influence of flow rate on theoretical plate number is shown in Figure 5.Most components under flow rate of 0.8-1.0mL min -1 presented high column efficiency.Considering the column efficiency and retention time (analytical speed) together, the optimal mobile phase flow rate was selected as 1.0 mL min -1 .

Selection of column temperature
Column temperature has a significant impact on the column performance, mobile phase viscosity and solvent solubility.With the increase in temperature, the mobile phase viscosity will be reduced, therefore, the mass transfer will be improved and the column pressure will be lowered.But high temperature will affect the separation degree between the components and easily produce bubbles in mobile phase.Considering the boiling point of dichloromethane (39.8 °C), four column temperatures at 23 (room temperature), 30, 32 and 34 °C were investigated.
With the increasing of column temperature, the retention time of all components showed a downward trend.And the bigger the n value was, the more obvious the downward trend presented.The influence of column temperature on separation degree was calculated and drawn as in Figure 6.It was found that the separation degrees between 12 components could reach 1.0 or more only under the column temperature of 30 °C.So the optimal column temperature was selected as 30 °C.

Selection of injection volume
The increasing of injection volume can improve sensitivity.But the excessive injection can cause wide  peak, tailed peak, even exceeding of column capacity and reduction of column life.Five injection volumes as 5, 10, 15, 20 and 25 μL were investigated in this study.The results showed that the peak area increased linearly with injection volume.But the separation degree of some components decreased below 1.0 with the injection volumes of 20 and 25 μL.Therefore, injection volume of 10 μL was decided by considering the sensitivity and the separation degree.

Chromatogram under optimal conditions and linear regression equations
Under the decided optimal chromatographic conditions (ternary mobile phase of isopropanol:n-hexane:dichloro methane with a gradient elution procedure; mobile phase flow rate of 1.0 mL min -1 ; column temperature of 30 °C; sample injection volume of 10.0 μL; HPLC-UV detection wavelength of 277 nm), the NP and 11 kinds of NPnEO were separated successfully within 35 min as shown in Figure 7.The separation of pure NP, mixed NP2EO and mixed NP5EO were also carried out to confirm the retention time of each component.According to the information (all components of the mixture are NPnEO with continuous n value) from Tokyo Chemical Industry Co. LTD and the principle (the bigger is the n value, the longer is the retention time of NPnEO), the relationship between retention time and the n value in Figure 7 was obtained.The concentration of each component in mixed standard solution was also decided by the area normalization method.
The used mixed standard solution was progressively diluted to 6 kinds of standard solutions (1, 2, 4, 8, 20 and 40 times) with different concentrations.After the chromatographic analysis of these diluted standard solutions, the standard curve of each component was obtained (see Figure 8).The linear regression equations, linear correlation coefficients and linear ranges are shown in Table 1.Except for NP11EO (97.2%), all other linear correlation coefficients achieved 99.9%.The limit of quantification (LOQ) values of the method were coincident with the smallest curve values in Table 1.
The precision experiments were repeated 11 times by measuring a certain concentration standard solution.The results are shown in Tables 2 and 3.Both retention time and peak areas were reproducible and all the RSD values (n = 0-11) obtained were satisfactory (< 5.0%).The stability of the standard solution was also investigated and the results showed that the standard solution could be stable for one week.

Wastewater sample determination
With the decided chromatographic method, the influent and effluent of the AOASP were analyzed.The results in Figure 9 indicated that the NP and NPnEOs in the wastewater samples were separated clearly and proved the effectiveness of this HPLC method.The concentrations       of NP and NPnEO in wastewater samples are shown in Table 4.It also proved the feasibility of AOASP in degrading NPnEOs.
A certain concentration of standard solution was added into the diluted influent to confirm the recovery rates.The detailed recovery rates information is shown in Table 5.The results showed that the average recovery rates of NP and NPnEO (n = 1-11) were 84.4-121.4%,meeting the needs of scientific analysis.It also proved the validation of the pretreatment method for the wastewater samples.

Conclusions
HPLC was selected for simultaneous determination of NP and NPnEO (n = 1-11) in wastewater samples from AOASP.The mobile phase parameters, column temperature and sample injection volume were optimized step by step.Under the optimized chromatographic conditions, the NP and 11 kinds of NPnEO were separated and determined successfully within 35 min.The linear correlation coefficients for the standard curves were from 0.9720 to 0.9999 and all the RSD values for precision degree were less than 5.0%.The average recoveries of NP and NPnEO (n = 1-11) for wastewater samples were 84.4-121.4% and achieved the scientific analysis requirement.The fast HPLC analytical method was proven successful and reliable, and could be used for relative NP and NPnEO determination.

Figure 5 .
Figure 5. Influence of flow rate on theoretical plate number.

Figure 6 .
Figure 6.Influence of column temperature on separation degree (R).

Figure 7 .
Figure 7. Chromatogram of mixed standard solution under the optimal conditions.The NP, mixed NP2EO and mixed NP5EO in mixed standard solution were 107.4,428.0 and 1968 μg mL -1 , respectively.

Table 1 .
Linear regression linear correlation coefficients and linear rangesComponentLinear equation Linear correlation coefficient Linear range a / (μg mL -1 quantification (LOQ) values of the method were coincident with the smallest curve values of linear range.

Table 2 .
Results of precision experiments (NP and NP1EO-NP5EO) a Relative standard deviation.

Table 4 .
Concentrations of NP and NPnEO in wastewater samples

Table 5 .
Recovery rates of influent