Determination of Biphenyl and Biphenyl Oxide in Aqueous Samples by Headspace Single Drop Microextraction Coupled to Gas Chromatography

Neste trabalho, bifenila e óxido de bifenila foram extraídos por microextração em headspace com única gota (HS-SDME) e analisados por cromatografia gasosa com detector de ionização em chama (GC-FID). A extração ocorreu a partir da ponta de uma microsseringa, pela suspensão de uma gota de tolueno, de 3,5 μL, como solvente extrator, contendo acetonaftona como padrão interno. O efeito de diferentes parâmetros como a natureza do solvente de extração, temperaturas da amostra e da microgota, velocidade de agitação, volumes da amostra e da microgota, força iônica e tempo de extração na eficiência da extração dos analitos foram investigados e otimizados. Sob condições ótimas, os limites de detecção (S/N = 3) da bifenila e óxido de bifenila foram 0,40 ± 0,03 e 0,30 ± 0,06 μg mL, respectivamente. Boas linearidades foram obtidas para ambos analitos com os coeficientes de correlação superiores a 0,997 e desvios-padrão relativos (R.S.D.) de 1 a 3%. As recuperações dos analitos a partir das amostras de água enriquecida foram próximas a 100%.


Introduction
One of the most common heat transfer fluids, dowtherm A, is a eutectic mixture containing 26.5% biphenyl and 73.5% biphenyl oxide. 1 Biphenyl is used as dye carrier for polyesters and feedstock, and a citrus fruit wrapping impregnate to reduce spoilage.It is especially used in the production process of alkylbiphenyls.Biphenyl oxide (BPO) is used in the production of emulsifiers, surfactants and textile dye labelling as a chemically reacted intermediate.
The main application of this mixture is heat transfer in the distillation tower and fatty acid production liner.This liquid has optimum heat coefficient with maximum service temperature up to 400 o C.There are many problems in fatty acid distillation tower when this liquid leaks from stocks to environment.The mixture is very harmful for human and ecological system.The concentrations of upper critical limit causes damage in liver and neurotic systems. 2 Thresholds limit values (TLV) are 0.2 g mL -1 for both of them in the surface waters. 3Conventional method for analyzing of this liquid is based on liquid-liquid extraction and gas chromatographic analysis, that is very time-consuming and uses much amount of organic solvent.The methods reported for measurement of biphenyl and biphenyl oxide are very limited and the solution is directly inject into the instruments in the all of them.Min and Wen 4 analysed the liquid by direct injection to gas chromatograph equipped with flame ionization detector (GC-FID) with the accuracy of 9 µg mL -1 .EPA method, 5 is based on direct injection of the mixture liquid to the gas chromatograph equipped with mass spectrometer detector (GC-MS) with the accuracy of 12 pg kg -1 .Moh et al. 6 have also used direct injection to the high performance liquid chromatography equipped with fluorescence detector (HPLC-FLD) with the accuracy of 2 µg mL - 1 .
8][9] Nonetheless, the necessity of reducing overall sample preparation time and quantities of organic solvents led to the development of several new extraction approaches, including solid-phase microextraction (SPME), [9][10][11] and solvent microextraction, [12][13][14] for the extraction of organic pollutants from environmental samples.Recently efforts have been focused on miniaturising the LLE procedure by greatly reducing the solvent to aqueous phase ratio, leading to the development of solvent microextraction methodologies.Single-drop microextraction (SDME) is one of these methodologies and involves extraction of organic contaminants from an aqueous donor solution into a microdrop of an organic acceptor solvent suspended on the tip of a microsyringe.SDME has the advantages of high extraction speed and extreme simplicity.There are two modes of sampling in measurement by SDME: direct single drop microextraction (direct-SDME) and headspace single drop microextraction (HS-SDME).Nowadays, both modes have been successfully used for the extraction of organic pollutants from a variety of matrices.It uses inexpensive apparatus and virtually eliminates solvent consumption.SDME has been successfully applied for the determination of alcohols, 15 nitroaromatics, 16,17 chlorobenzenes, 18 drugs, 19,20 volatile organic compounds (VOCs), 21 polycyclic aromatic hydrocarbons (PAHs), 22 aliphatic amines, 23 BTEX, 24 and also for the screening of pesticides [25][26][27] in water samples.
To the best of our knowledge, there are any reports that used HS-SDME for the extraction of biphenyl and biphenyl oxide.This work used HS-SDME method for the extraction of biphenyl and biphenyl oxide from aqueous samples.The method provided a sensitive and easy-to-use tool for the environmental monitoring of these contaminants.For obtaining the optimum extraction conditions, different parameters affecting the extraction efficiency were studied and optimised.

Reagents and materials
Biphenyl, biphenyl oxide, acetonaphton, 1-butanol, 1-octanol, n-hexane, toluene, acetone, cyclohexane, isopropyl alcohol, benzyl alcohol, dihexyl ether, reagent grade sodium chloride and HPLC grade methanol were purchased from Merck.The stock standard solutions of biphenyl, biphenyl oxide, acetonaphton (1000 mg L -1 ) were prepared in methanol.They were stored and refrigerated at 4 o C. Biphenyl and biphenyl oxide stock standard solutions were diluted with methanol to prepare a mixed stock solution of analytes with concentration of 100 mg L -1 .Working standard solutions were freshly prepared by diluting the mixed standard solution with distilled water to the required concentrations.A solution of acetonaphton as internal standard (IS) with 15 mg L -1 concentration was prepared in toluene.This solution was used as extracting phase.

Apparatus
A 10-µL Hamilton microsyringe (Hamilton, Bonaduz, Switzerland) model 701RN with a bevel needle tip (length 5.1 cm, ID 0.013 cm, bevel 22) was used for the extraction and injection procedures.Stirring the solution was carried out with a magnetic stirrer (Heidolph MR 3001 K) and a 8 mm × 1.5 mm stirring bar.Two circulating water bathes were used for adjusting the temperatures of syringe needle and sample solutions with accuracy of ± 0.01 o C. For HS-SDME procedure, a glass cell fabricated with stainless steel inner tube (1.16 inch o.d.) was used for controlling the sample temperature.In order to reach a temperature very close to the temperature of cooling bath, the internal surface of the inner tube was contacted to the external surface of the microsyringe needle 28 (Figure 1).
Separation and quantification of biphenyl and biphenyl oxide analytes were carried out by using a Shimadzu-14 B gas chromatograph equipped with a flame ionization detector and a DB-5 (5% biphenyl + 95% ploydimethylsiloxane) fused-silica capillary column with a 25 m × 0.33 mm I.D. and 0.5 µm film thickness (Shimadzu).The injection port and detector were operated at 275 and 285 o C, respectively.The GC split valve was open and helium was used as carrier gas to give a 4 mL min -1 column flow and 5 mL min -1 split line flow.The detector gases flow rates were 300 mL min -1 for air and 30 mL min -1 for hydrogen.The column was held at 100 o C for 1 min, increased to final temperature of 260 o C at a ramp of 10 o C min -1 and then held for 5 min in this temperature.

HS-SDME procedure
A fixed concentration of acetonaphton (15 mg L -1 ) as internal standard was prepared in toluene as extracting solvent.A 6 mL aliquot of the mixture was placed in the 14 mL vial with a PTFE-silicon septum (Supelco).The Hamilton microsyringe was completely washed with methanol and acetone, respectively.After drying the syringe, it was rinsed and primed at least seven times with the solvent/internal standard.After uptake of 5 µL of extractant solvent, the needle of the syringe is then inserted into the internal tube of the two compartment cell above the extraction vial (that was being cooled with circulating antifreeze), pierced the vial septum and then was clamped.By applying the two compartment cell, the needle tip was located in a constant position in the headspace of the solution.For starting the extraction, the syringe plunger was depressed and a microdrop of extraction solvent was suspended from the needle tip.After extracting for a prescribed time, the plunger was withdrawn and the microdrop was retracted back into the syringe.The needle was consequently removed from the vial and its contents were injected into the GC for the analysis.Finally, the analytical signal was shown as the relative peak area of the analyte to the internal standard.

Results and Discussion
The initial objective was to optimize the HS-SDME sampling conditions and to fix the parametric values for the extraction of biphenyl and biphenyl oxide.The dynamic characteristics of the microextraction process are closely related to the mass transfer of the analytes from the aqueous to the organic phase.Intrinsically, the SDME process is driven by the difference gradient of concentration between aqueous and organic phases.There were several parameters which determine the mass transfer such as the extraction solvent, drop and sample volume, ionic strength, stirring rate, extraction time and temperature.These parameters were chosen to optimize the extraction efficiency via a univariate optimization approach.Conditions for HS-SDME were tested using water solution of 25 µg L -1 for each analyte.To obtain optimized extraction conditions, the relative peak area of analyte to the internal standard (acetonaphton) was used in the GC-FID analysis of extracts.

Selection of organic solvent
The selection of an appropriate extraction solvent is of great importance for the optimization of HSME process.Theoretical considerations of HSME show that mass transfer of the analytes from the sample to the microdrop continues until thermodynamic equilibrium is attained or the extraction is stopped.The principle "like dissolves like" can become the basis of the solvent selection.Different solvents were tested to find a suitable solvent that has high extraction efficiency for the target compounds, low volatility, less toxicity and satisfactory chromatographic resolution for the analytes.Low volatility is helpful to keep the solvent microdrop at the tip of a microsyringe needle sustainable over the extraction time period.In order to determine which solvent would be optimal for the extraction of biphenyl and biphenyl oxide, several solvents including 1-butanol, 1-octanol, n-hexane, toluene, cyclohexane, isopropyl alcohol, benzyl alcohol were examined.Among different solvents examined benzyl alcohol gave the best extraction efficiency and was kept stable at the tip of syringe over the extraction period, but their chromatographic peaks overlapped with some of the analyte peaks (Figure 2).
After a detailed peak areas obtained by other solvents for the two compounds (Figure 3), toluene was found to be optimal and finally chosen as the extraction solvent for the HS-SDME analysis.For all quantification experiments, fixed concentration of acetonaphton (15 mg L -1 ) as internal standard was prepared in toluene as extracting solvent.

Microsyringe needle temperature
Variation of the extraction efficiency with microsyringe needle temperature in the range of 0 to 10 °C was shown in Figure 4.According to this observation, the microdrop/ sample distribution coefficient decreases with increasing enhancement in the sample matrix-extraction phase distribution constant, associated with the temperature gap present in the system, can be described by the equation 1, 29 (1) where K T = C e (T e )/C h (T h ) is the distribution constant of the analyte between the cold extraction phase (i.e., the microdrop having temperature T e ) and hot headspace at temperature T h .C p is the constant-pressure heat capacity of the analyte, DT = T h -T e , and K 0 is the organic drop/ headspace distribution constant of the analyte when both microdrop and headspace are at temperature T e .Finally, C e and C h are the concentrations of the solute in the microdrop and headspace, respectively.
The decreased needle temperature leads to the condensation of analyte on the cooled microdrop and thus, increases the extraction efficiency.The main reason is that the process of analyte absorption in the microdrop is exothermic and the partition coefficient, K mh , is temperature dependent. 30Hence, further extractions were performed at a microsyringe needle temperature of 0 °C.

Sample temperature
Usually increasing sample temperature increases the evaporation and transportation of analytes from the sample matrix to the headspace and will result in improved extraction efficiency.The influence of sample temperature on the extraction efficiency of the system was studied from 10 to 60 °C and results are also shown in Figure 5. On    microdrop temperature, which results in the decreased sensitivity of the extraction process.This additional the other hand, increasing the temperature of the solution above 45 °C can cause drop damage and evaporation of microdrop.Therefore, the extraction temperature of 45 °C was considered as the optimum temperature in the following experiments.

Solvent drop volume
The HS-SDME theory indicates a dynamic equilibrium established between the concentration of the analytes in the headspace and organic solvent drop.The amount of analyte, n, extracted by the microdrop is related to the volume of the drop, and the sensitivity improves when the volume of the drop increases (equation 2), 30 (2) where k odw and k hs are the organic drop-water (sample) and the headspace-water distribution constants, respectively; C 0 the initial concentration of the analyte in the matrix; and V d , V s and V h are the volumes of the drop, sample, and headspace, respectively.In the present study, solvent volumes were tested in the range of 4 to 8 µL and the results are shown in Figure 6.
The highest extraction efficiency was obtained at the drop volume of 7.0 µL.Therefore, the drop volume of 7.0 µL was selected for the subsequent experiments.

Sample volumes
According to the equation 2, increasing sample volume and consequently a decrease in headspace volume leads to an increase of the analytes concentrations in the headspace, and thus enhances the extraction efficiency, which improves the sensitivity.In order to study the effect of the sample volume on the extraction efficiency, the volumes of samples were from 5 to 50 mL by using 60 vials.The results showed that the largest analytical response was obtained at sample volume of 50 mL (Figure 7).

Stirring rate
Sampling agitation enhances extraction efficiency and reduces extraction time, because the equilibrium between the aqueous and vapor phases can be achieved more rapidly and the diffusion of the analytes toward the microdrop can be enhanced. 30In this work, different stirring rates (300, 400, 600, 800, 1000 rpm) with a 8 mm stirring bar were studied.As shown in Figure 8, the peak areas of both analytes increase by increasing the stirring rate up to 800 rpm.Faster stirring rates were avoided   because of the dislodgement of the organic drop from the needle.Hence, a stirring rate of 800 rpm was chosen for further studies.

Ionic strength
It was of interest to examine the influence of salt addition on the extraction efficiency.Therefore the ionic strength of solutions was modified by increasing NaCl concentration from 0 to 5.7 mol L -1 in the water sample.The extraction efficiency improved with an increasing salt concentration to a maximum value of 3 mol L -1 .Results were shown in Figure 9.It is evident that the addition of NaCl to 3 mol L -1 promotes the transport of the analytes to the headspace and hence to the extraction drop.Decreasing in the extraction efficiency was observed at higher salt concentration of 3 mol L -1 .It is due to this fact that NaCl dissolved in water might have changed the physical properties of the Nerst diffusion film and decreases dielectric constant of water.It increases stability of analytes and resulting in less efficient extraction.Thus, other measurements were carried out at a NaCl concentration of 3 mol L -1 .

Extraction time
The optimum extraction time was determined by varying the exposition time of the microdrop in the headspace of

Performance of the HS-SDME method
Under optimum conditions, linearity was observed over the concentration range of 5-150 µg L -1 for both target compounds.The corresponding regression equation, correlation coefficient (R 2 ), limits of quantitation (LOQ), dynamic linear range (DLR), limit of detection (LOD) and the enrichment factor (E e ) were calculated and summarized in Table 1.The limits of detection (LOD) of all target compounds were calculated by comparing the signal-tonoise (S/N) ratio of the lowest detectable concentration to a S/N ratio of 3. The limits of quantitation (LOQ), were calculated as concentrations giving signal-to-noise ratio = 10.In order to calculate the enrichment factor of each analyte, three replicate extractions were performed at optimal conditions from aqueous solution.The enrichment factor, 31 Ee, defined as the ratio of the equilibrium concentration of biphenyl and biphenyl oxide in the organic phase to the original concentration of biphenyl and biphenyl oxide in the aqueous phase, were 84 and 87.

Matrix effects assessment and application to real samples
To demonstrate the performance of the proposed HS-SDME method, four real water samples including; the water sample output from distillation tower, the water sample input to the condenser chamber with a distance of 300 meters from the distillation tower, the water sample which is 350 meters away from the distillation chamber and waste water of Paxan Co. (Tehran, Iran).The extraction was repeated for each sample.Relative recoveries and precision were calculated and listed in Table 3.As a result, acceptable recoveries (96.9-112.1%)and R.S.D. values (0.3-1.9%) were obtained for the two analytes in the tested water samples.These results demonstrate that deferent water matrix had no effect on the HS-SDME.

Conclusions
Simplicity and ease of use, good analytical precision and accuracy, short overall sample preparation time and low cost are some of the advantages of proposed liquid phase microextraction method.The method showed low R.S.D. and LOD values, wide dynamic linear ranges, high enrichment factors and good recoveries for target compounds.The proposed procedures were also successfully applied with acceptable sensitivity and limit of detection for the determination of biphenyl and biphenyl oxide in real water samples.

Figure 4 .
Figure 4. Effect of microsyringe needle temperature on the extraction efficiency of HS-SDME for biphenyl and biphenyl oxide.Other experimental conditions are as follows: concentration level at 25 µg L -1 ; 300 rpm stirring rate; 5 µL drop volume; 10 mL sample volume; sample temperature: 25 °C; 0% NaCl; 5 min extraction time.

Figure 5 .
Figure 5.Effect of sample temperature on the extraction efficiency of HS-SDME for biphenyl and biphenyl oxide.Other experimental conditions are as follows: concentration level at 25 µg L -1 ; 300 rpm stirring rate; 5 µL drop volume; 10 mL sample volume; microsyringe needle temperature, 0 °C; 0% NaCl; 5 min extraction time.

Figure 6 .
Figure 6.Effect of sample drop volume on the extraction efficiency of HS-SDME for biphenyl and biphenyl oxide.Other experimental conditions are as follows: concentration level at 25 µg L -1 ; 300 rpm stirring rate; 10 mL sample volume; microsyringe needle temperature, 0 °C; sample temperature, 45 °C; 0% NaCl; 5 min extraction time.

Table 1 .
Limit of detections and quantitation, regression equations, correlation coefficients, dynamic linear ranges, R.S.D., and enrichment factors for HS-SDME of biphenyl and biphenyl oxide

Table 2 .
Comparision of performance data of Headspace SDME, in-loop SPME-LC, dispersive liquid-liquid microextraction based on solidification of floating organic droplet method (DLLME-SFO) and dispersive LLME

Table 2
compares the performance data of proposed methods and other microextraction methods.The table shows that the proposed method posses reasonable performance data in compared with other microextraction methods.