Determination of N-Octanol/Water Partition Coefficient for DDT-Related

Chemosphere 83 (2011) 131–136

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Determination of n-octanol/water partition coefﬁcient for DDT-related compounds by RP-HPLC with a novel dual-point retention time correction ⇑ ⇑ Shu-ying Han a, Jun-qin Qiao a, Yun-yang Zhang a, Li-li Yang b, Hong-zhen Lian a, , Xin Ge a, , Hong-yuan Chen a a Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), School of Chemistry & Chemical Engineering and Center of Materials Analysis, Nanjing University, 22 Hankou Road, Nanjing 210093, China b Nanjing Environmental Monitoring Center, 175 Huju Road, Nanjing 210013, China article info abstract

Article history: n-Octanol/water partition coefﬁcients (P) for DDTs and dicofol were determined by reversed-phase high Received 22 June 2010 performance liquid chromatography (RP-HPLC) on a C18 column using methanol–water mixture as Received in revised form 18 December 2010 mobile phase. A dual-point retention time correction (DP-RTC) was proposed to rectify chromatographic Accepted 5 January 2011 retention time (tR) shift resulted from stationary phase aging. Based on this correction, the relationship

between log P and log kw, the logarithm of the retention factor extrapolated to pure water, was investigated for a set of 12 benzene homologues and DDT-related compounds with reliable experimental P as Keywords: model compounds. A linear regression log P = (1.10 ± 0.04) log k – (0.60 ± 0.17) was established with n-Octanol/water partition coefficient (P) w correlation coefficient R2 of 0.988, cross-validated correlation coefficient R2 of 0.983 and standard devi- RP-HPLC cv Dual-point retention time correction (DP- ation (SD) of 0.156. This model was further validated using four verification compounds, naphthalene, 0 RTC) biphenyl, 2,2-bis(4-chlorophenyl)-1,1-dichloroethane (p,p -DDD) and 2,2-bis(4-chlorophenyl)-1,1- DDTs dichloroethene (p,p0-DDE) with similar structure to DDT. The RP-HPLC-determined P values showed good Dicofol consistency with shake-flask (SFM) or slow-stirring (SSM) results, especially for highly hydrophobic POPs compounds with log P in the range of 4–7. Then, the P values for five DDT-related compounds, 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1,1-trichloroethane (o,p0-DDT), 2-(2-chlorophenyl)-2-(4-chlorophenyl)- 1,1-dichloroethane (o,p0-DDD), 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene (o,p0-DDE), and 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol) and its main degradation product 4,40-dichlorobenzophenone (p,p0-DBP) were evaluated by the improved RP-HPLC method for the first time. The excellent precision with SD less than 0.03 proved that the novel DP-RTC protocol can significantly increases the determination accuracy and reliability of P by RP-HPLC. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction et al., 1974; Platts et al., 2006). Therefore, there is a continuing de- mand for acquiring accurate and reliable P values. n-Octanol/water partition coefficient (P) provides direct infor- By now, a variety of methods have been in use to obtain P, mation on hydrophobicity that describes the tendency of distribu- which are divided into two major classes: experimental measure- tion of a solute from aqueous phase into organic constituents of ment and theoretical calculation. Traditional experimental meth- environmental compartments (Kenaga, 1980; Karickhoff, 1981; ods are shake-flask method (SFM) (OECD, 1981) and slow-stirring Bodor et al., 1989), and even into biological membranes (Kubinyi, method (SSM) (Brooke et al., 1986). Although basically correct, 1979; Sangster, 1989; Bechalany et al., 1991). This has made it SFM and SSM have some drawbacks concerning the consumption one of the most commonly reported physico-chemical properties of time and labor. Moreover, it is unreliable by using SFM for sub- of drugs, pesticides and other chemicals (Leo, 1991; Lambert, stances having high lipophilicity (log P > 5) due to the formation of 1993). Furthermore, P is frequently used to predict water solubil- emulsions between octanol and water phases during the shaking ity, soil (sediment)/water partition coefficient, bioconcentration (Finizio et al., 1997). A large number of theoretical methods also factor and nonreactive toxicity, etc. (Gobas et al., 1988; Neely have been developed for calculation of P. Among them, the most fa- mous one is fragmental constant approach proposed by Leo et al. ⇑ (1971). However, theoretical calculation is inclined to be inaccu- Corresponding authors. Tel.: +86 25 83686075; fax: +86 25 83325180 (H.-z. rate for compounds possessing several functional groups with Lian), tel.: +86 25 83592466; fax: +86 25 83592723 (X. Ge). E-mail addresses: [email protected] (H.-z. Lian), [email protected] (X. Ge). interactions not sufficiently defined. For relatively complex

molecules, such as some pesticides, the breakdown of the molecule where tR and tR represent the retention time and average retention can produce, in a few cases, large or unusual fragments for which time, respectively, of oligonucleotides. In fact, it is a dual-point fragmental values are not available in the list provided by the (DP)-RTC method. The essence of this DP-RTC is considered as rec- method or in the software library (Finizio et al., 1997). Reversed- tifying tR measured at different column situations to the one ob- phase high performance liquid chromatography (RP-HPLC) (Garst tained on an ‘‘imaginary average column’’ that is expressed as and Wilson, 1984) is another commonly adopted method for deter- tR,normalized. However, this normalization procedure have to be taken mining P for a wide range of compounds (OECD, 1989; Kurz and out after all experiments have been ﬁnished for the acquisition of

Ballschmiter; 1999; Paschke et al., 2001), which is proved to show final tR data only by a selected mobile phase composition at which more reliable results than theoretical calculation (Finizio et al., all investigated oligonucleotides were completely separated for 1997). It profits from the relationship between the chromato- retention behavior study. In this present work, we proposed a novel graphic retention on a reversed-phase column and P of analytes. DP-RTC protocol to obtain reliable tR by assigning a real LC column Compared to SFM and SSM, RP-HPLC is more rapid and easy-to- used on a certain day (usually the day a column first used) as ‘‘stan- operate because it does not require any quantification of concen- dard column’’ instead of ‘‘imaginary average column’’ suggested by tration, and only retention time (tR) needs to be measured. Notice- Kohlbacher et al. Two specific standard compounds are employed as ably, determination of P by RP-HPLC is highly dependent on ‘‘anchor compounds’’. This normalization is able to rectify tR ob- accurate solute retention. Even though retention factor (k) is re- tained every day to that obtained on the certain day, thus reflects lated to P, for a given solute and a given stationary phase, it will de- the truest retention behavior of analytes. pend on the composition of the hydroorganic mobile phase Persistent organic pollutants (POPs) have drawn more and more

(Sangster, 1997). Therefore, kw, the hypothetical retention factor attentions because of their highly bioaccumulative, carcinogenic, corresponding to 100% water, instead of k at a specific organic mutagenic and environmental endocrine disruptor effects. P is a modifier content, is employed (Snyder et al., 1979; Braumann, key physico-chemical property of POPs not only for evaluating their 1986). In this case, the retention is independent of any organic fate and distribution in the environment (Cohen et al., 1990; modifier effects, and the determined P is more similar to SFM/ Mackay, 1991), but also for assessing their accumulation and toxic- SSM-measured one. Some papers pointed out that the reliability ity in organism tissues (Mackay, 1982;USEPA, 1992). Nevertheless, for P determination of RP-HPLC is in the range of log P 0–6 (OECD, till now P data reported for POPs are still deficient and sometimes 1989). Problematic with more hydrophobic substances is the long- incorrect, and even discrepant each other for a compound. This is er retention during chromatographic elution, which may cause because their strong hydrophobicity is unsuitable for SFM measure- uncertainty of tR owing to peak broadening. Gradient elution has ment, or because their high structure complexity is difficult for soft- been tried to solve this problem (Krass et al., 1997; Verbruggen ware calculation. Therefore, RP-HPLC has been considered as a et al., 1999), but it actually takes more run time for system equilib- promising alternative for estimating P values of POPs in recent years rium before next injection, and normally gives poorer reproducibil- (Ayouni et al., 2005; Demare et al., 2007). DDT, one of the most well- ity in tR measurement than isocratic elution. In addition, whatever known synthetic organochlorine pesticides, belongs to POPs. The isocratic or gradient mode is used, some P data obtained by RP- term DDTs (total DDT) is often used to refer to the sum of all DDT HPLC still have large discrepancy with those of SFM/SSM. (dichlorodiphenyltrichloroethane), DDD (dichlorodiphenyldichlo- As we know, silica-based stationary phase collapses unavoid- roethane) and DDE (dichlorodiphenyldichloroethylene), as well as ably by continual flushing of aqueous mobile phase during the their isomers. DDE and DDD are also the major metabolites and course of usage. This column aging causes shift of tR for an analyte breakdown products of DDT in the environment. In addition, under the same chromatographic conditions in different measuring 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol) is an orga- periods. Therefore, tR should be rectified to reduce this deviation nochlorine miticide that is prepared from DDT and chemically especially in the study of retention behaviors. The most common related to DDT. Dicofol is easily converted to DBP (dichlorobenzo- solution is to simply employ t0 of a dead time marker at each elu- phenone). DDTs is found to exist extensively in soil and water as a tion condition to rectify tR, which actually is a single-point reten- result of its abuse beforetime and manufacture of dicofol nowadays. tion time correction (SP-RTC). SP-RTC means large random error As far as our knowledge goes, there have been no reliable directly because t0 is too short to be recorded accurately. A few multi-point experimental P values reported for some DDT-related compounds (MP)-RTC methods also have been tried to improve measurement yet. In this work, P values of 2-(2-chlorophenyl)-2-(4-chloro- 0 precision by standardizing tR. A sophisticated standardization pro- phenyl)-1,1,1-trichloroethane (o,p -DDT), 2-(2-chlorophenyl)-2- cedure was developed by Sapirstein et al. (1989) using several se- (4-chlorophenyl)-1,1-dichloroethane (o,p0-DDD), 2-(2-chloro- 0 lected peaks from a special protein sample as tR standards which phenyl)-2-(4-chlorophenyl)-1,1-dichloroethene (o,p -DDE), dicofol 0 0 were analyzed before and after the sample of interest. tR of these and 4,4 -dichlorobenzophenone (p,p -DBP) are determined by peaks were then used as ‘‘anchor points’’ in a piecewise calibration RP-HPLC combined with DP-RTC. This DP-RTC-RP-HPLC method algorithm to normalize the chromatograms of samples run in the facilitates a reliable prediction of P for DDTs and dicofol. interval between two of the standards. But it is assumed that in this experiment, LC conditions should be exactly same for standards and samples. Petritis et al. (2003) proposed a Genetic Algo- 2. Materials and methods rithm for normalization to optimize two variables of a linear equation, y = ax + b. The variable a normalizes gradient slope and 2.1. Materials b normalizes LC run start time (dead volume, delay time, etc.). The optimization of a and b is performed for each separation, and Methanol (HPLC grade) was purchased from Merck (Darmstadt, the normalized tR falls into a 0–1 range, hence this method could Germany). Purified water (Wahaha Group Ltd., Hangzhou, China) not offer real tR values of analytes. Kohlbacher et al. (2006) sug- was used throughout the experiment. Table 1 lists compounds gested another normalization method in DNA retention behavior investigated in this experiment, which were divided into three study. Two oligonucleotides (dC)14 and (dT)26 were co-injected as sets: training, validation and determination sets. These compounds internal standards to normalize tR via the following equation: were with the purity of 98% or greater checked by RP-HPLC, and then used without further purification. Individual stock solution tR dT tR dC t ¼ðt t Þ ð Þ26 ð Þ14 þ t ð1Þ (ca. 1.0 mg mL1) was prepared in methanol and stored in refriger- R;normalized R;measured RðdCÞ14 t t RðdCÞ14 RðdTÞ26 RðdCÞ14 ator before use. S.-y. Han et al. / Chemosphere 83 (2011) 131–136 133

Table 1 compounds, were chosen as ‘‘anchor compounds’’ to obtain slope Investigated compounds. m in the following experiments every day. Compounds Log Pa Reference Clog Pb All investigated compounds were eluted at a minimum of four Model compounds (training set) isocratic methanol–water ratios according to their hydrophobicity. Anisole 2.04 Rogers and Cammarata (1969) 2.13 ± 0.20 tR was recorded at each methanol–water ratio along with the dead Benzyl chloride 2.30 Sangster (1989) 2.49 ± 0.21 time t0 determined using sodium nitrate, then rectified by SP- and Toluene 2.61 El Tayar et al. (1984) 2.68 ± 0.17 DP-RTC, respectively. The k value was calculated according to the Ethylbenzene 3.15 Sangster (1989) 3.21 ± 0.17 equation k =(t –t )/t . For each solute, the logarithm of k was plot- 2-Methylnaphthalene 4.00 Sangster (1989) 3.77 ± 0.17 R 0 0 1,2,4-Trichlorobenzene 4.02 Chiou (1985) 3.82 ± 0.32 ted against the volume fraction of methanol, and log kw of the ana- 1,2,3-Trichlorobenzene 4.05 Watarai et al. (1982) 3.77 ± 0.32 lyte was subsequently obtained by extrapolation of log k to pure 1,3,5-Trichlorobenzene 4.19 De Bruijn et al. (1989) 4.04 ± 0.31 water via Snyder-Soczewinski equation (Snyder et al., 1979). Then Hexamethylbenzene 4.75 Sangster (1989) 4.98 ± 0.20 the log P–log kw regression equation of 12 model compounds with Pentachlorobenzene 5.17 Watarai et al. (1982) 4.60 ± 0.35 Hexachlorobenzene 5.73 De Bruijn et al. (1989) 4.89 ± 0.36 reliable SFM P data was established. Cross-validation, usually char- 2 2 0 p,p -DDT 6.36 Karickhoff et al. (1979) 5.92 ± 0.40 acterized by Rcross-validated (Rcv), is a practical and reliable method for Verification compounds (validation set) inspecting the significance of a model. In this work, leave-one-out Naphthalene 3.01 Rogers and Cammarata (1969) 3.45 ± 0.16 (LOO) procedure was used as internal validation to examine the Biphenyl 3.95 Hansch and Leo (1979) 3.98 ± 0.23 reliability and prediction capability of the model. R2 was calcu- p,p0-DDD 5.69 Hansch and Leo (1979) 5.39 ± 0.36 cv p,p0-DDE 6.96 De Bruijn et al. (1989) 6.37 ± 0.37 lated by estimating in turn the log P value for one of model com- Sample compounds (determination set) pounds from the regression equation calculated from the data o,p0-DDT – 5.92 ± 0.40 for all other model compounds. The statistical analysis was accom- 0 o,p -DDD – 5.39 ± 0.36 plished by SPSS V16.0.0 (SPSS Inc., Chicago, Illinois, USA) and MAT- o,p0-DDE – 6.22 ± 0.37 p,p0-DBP – 4.62 ± 0.33 LAB Software V7.10.0 (R2010.a) (The MathWorks, Inc., Natick, MA, Dicofol – 5.74 ± 0.43 USA). In addition, in order to assess the true predictive potential of the model, the regression was inspected by an external validation. a SFM data were adopted, except p,p0-DDE for which SSM data was adopted. Four verification compounds (naphthalene, biphenyl and p,p0-DDD b Calculated by Advanced Chemistry Development (ACD/Labs) Software V8.14 for 0 Solaris (1994–2007 ACD/Labs). with SFM P, and p,p -DDE with SSM P) were employed to confirm the consistency between DP-RTC-RP-HPLC-determined log P in this work and SFM/SSM-measured ones. Finally, the validated 2.2. Apparatus 0 0 log P–log kw model was applied to evaluate P of o,p -DDT, o,p - DDD, o,p0-DDE, dicofol and p,p0-DBP through their individual A LabTech 600 LC (Lab-Tech Instru., Beijing, China) was em- extrapolated log k in RP-HPLC rectified by the improved DP-RTC ployed consisting of a Rheodyne 7725i injector valve equipped w method. The whole experimental procedure was repeated three with a 10-lL loop (Cotati, CA, USA), a HB-230A incubator (Hanbon times. Sci. Tech., Huai’an, China) and a UV–vis 600 Detector (Lab-Tech) set at the respective optimum absorption wavelength for each eluted compound. The chromatographic column used was a Kromasil 3. Results and discussion C18,5lm, 150 mm 4.6 mm i.d. (Hanbon) maintained at 30 °C. The mobile phase was methanol–water. Data acquisition and pro- 3.1. Retention time correction (RTC) cessing were performed on a LC workstation 2006 (Lab-Tech). All experimental retention times were obtained by averaging the re- The tR normalization proposed by Kohlbacher et al. (2006) was 1 sults of at least two independent injections at 1.0 mL min mobile actually a kind of internal standard method for RTC. The feasibility phase flow rate. of that method was just because they used only a specific mobile phase condition under which all the investigated compounds could 2.3. Procedure separate completely, and only a tR value was required for each compound. However, in our work, P measurement was based on

The linear correlation of tR values acquired on different RP-HPLC the linear log P–log kw relationship. Log kw of the investigated com- columns for a set of compounds under an identical eluent has been pound was obtained by extrapolation of log k under at least four proved in many works (Jaitly et al., 2006; May et al., 2007). In the ratios of methanol–water mixture to pure water. It is impossible circumstances, there should be better linearity between tR values to simultaneously separate all of these compounds in a run under on one column for a set of compounds under an identical eluent each elution condition. Unavoidable overlapping of peaks will on different days. tR of a solute measured on an aged column on make inaccurate record of retention times. Alternatively, single some day could be rectified to the one equivalent to be obtained sample injection was chosen in our experiments, so the proposed on ‘‘standard column’’ by a calibration equation. In this experi- DP-RTC is an external standard method for qualitative analysis ment, tR of 12 model compounds including DDT-related com- by retention time in a sense. The precision of this RP-HPLC method pounds were collected at the mobile phase of 80% methanol by is governed by the reproducibility of tR measurement. The compar- volume on two different days, of which one is the day ‘‘standard ison of log kw values between SP- and DP-RTC are shown in Table 2. column’’ first used, then these two groups of vectors were fitted The relative standard deviations (RSDs) of log kw measured over a to the linear equation: period of several months were ranged from 0.12% to 3.27% (SP- RTC) and from 0.02% to 0.50% (DP-RTC), respectively, indicating the better reproducibility by using DP-RTC than SP-RTC. tR;measured ¼ mtR;standard þ n ð2Þ

3.2. Validation of DP-RTC-RP-HPLC method The correlation coefﬁcient (R2 P 0.999) of the regression conﬁrmed that this linearity also exists in retention times of DDT-related com- The correlation between log P and log kw derived from DP-RTC, pounds. Based on this excellent linearity, anisole and hexamethyl- as well as the LOO cross-validation result for 12 model compounds benzene, having acceptable wide tR interval among model of training set was given: 134 S.-y. Han et al. / Chemosphere 83 (2011) 131–136

Table 2

The log kw values of investigated compounds (n = 3).

Compounds SP-RTC DP-RTC

Log kw Mean log kw RSD (%) Log kw Mean log kw RSD (%) Model compounds Anisole 2.332, 2.359, 2.357 2.349 0.64 2.362, 2.357, 2.355 2.358 0.15 Benzyl chloride 2.846, 2.882, 2.885 2.871 0.76 2.902, 2.885, 2.887 2.891 0.32 Toluene 2.943, 2.932, 2.934 2.936 0.20 2.964, 2.941, 2.943 2.949 0.43 Ethylbenzene 3.468, 3.470, 3.477 3.472 0.14 3.482, 3.484, 3.489 3.485 0.10 2-Methylnaphthalene 3.947, 3.868, 3.859 3.891 1.24 3.973, 3.984, 3.973 3.977 0.16 1,2,4-Trichlorobenzene 4.057, 3.984, 3.989 4.010 1.02 4.085, 4.081, 4.086 4.084 0.07 1,2,3-Trichlorobenzene 3.987, 3.911, 3.914 3.937 1.09 4.015, 4.008, 4.011 4.011 0.09 1,3,5-Trichlorobenzene 4.257, 4.195, 4.189 4.214 0.89 4.280, 4.288, 4.282 4.283 0.10 Hexamethylbenzene 4.916, 4.771, 4.774 4.820 1.72 4.970, 4.962, 4.965 4.966 0.08 Pentachlorobenzene 5.117, 4.987, 4.988 5.031 1.49 5.168, 5.177, 5.178 5.174 0.11 Hexachlorobenzene 5.807, 5.545, 5.578 5.643 2.53 5.854, 5.817, 5.827 5.833 0.33 p,p0-DDT 6.265, 6.187, 6.189 6.214 0.72 6.325, 6.376, 6.378 6.360 0.47 Veriﬁcation compounds Naphthalene 3.164, 3.168, 3.169 3.167 0.08 3.318, 3.318, 3.317 3.318 0.02 Biphenyl 3.785, 3.788, 3.783 3.785 0.07 3.988, 3.989, 3.983 3.987 0.08 p,p0-DDD 5.711, 5.403, 5.396 5.503 3.27 5.589, 5.549, 5.548 5.562 0.42 p,p0-DDE 6.762, 6.666, 6.720 6.716 0.72 6.746, 6.781, 6.786 6.771 0.32 Sample compounds o,p0-DDT 6.580, 6.483, 6.485 6.516 0.85 6.603, 6.660, 6.662 6.642 0.50 o,p0-DDD 5.359, 5.368, 5.371 5.366 0.12 5.467, 5.476, 5.482 5.475 0.14 o,p0-DDE 5.716, 5.722, 5.719 5.719 0.05 5.842, 5.852, 5.849 5.848 0.12 p,p0-DBP 3.991, 4.009, 4.015 4.005 0.31 4.104, 4.115, 4.125 4.115 0.26 Dicofol 6.003, 5.916, 5.918 5.946 0.84 6.011, 6.038, 6.040 6.030 0.27

log P ¼ð1:10 0:04Þ log kw ð0:60 0:17Þð3Þ slopes of two regressions are both close to 1, revealing that chromatographic retention and n-octanol/water partitioning are 2 2 where R = 0.988, Rcv = 0.983, SD = 0.156, and F = 806.6. The linear approximately homoenergetic processes. Moreover, the regression regression model with satisfactory internal validation result shown by DP-RTC is superior to that by SP-RTC in the suitability for sim- in Eq. (3) and Fig. 1 indicates that the model is able to correlate log P ulating partitioning between n-octanol and water by RP-HPLC. to log kw with high statistical signiﬁcance. Meanwhile, the regres- Satisfactory external validation result was also given for four sion relating log P with SP-RTC derived log kw was also obtained: DDT-related compounds. The deviations (60.2 log units) (Table 3) indicate that this DP-RTC-RP-HPLC method has a comparable level of precision to SFM/SSM for determining log P of DDTs. Table 3 log P ¼ð1:16 0:04Þ log k ð0:74 0:18Þð4Þ w also summarizes other RP-HPLC log P values collected from literatures for these four chemicals. It must be noted that not only these 2 2 where R = 0.986, Rcv = 0.982, SD = 0.160, and F = 764.9. values have obvious differences between each other, but also all of As claimed by Minick et al. (1989), Liu et al. (2005) and Giaginis them have signiﬁcant negative deviations compared to SFM/SSM et al. (2007), the slope of an equation correlating log P (n-octanol/ data, and these deviations increase with the hydrophobicity of 0 0 water partitioning) and log kw (chromatographic retention) is an compounds. When log P > 5, as p,p -DDD and p,p -DDE, the estimate of how closely the free energies of the processes compare, reported RP-HPLC-determined log P values even have poorer accu- and a unit slope in such a plot implies that these two processes are racy than ACD/Lab-calculated ones. This phenomenon is in accord homoenergetic. It can be observed from Eqs. (3) and (4) that the with the statement of Finizio et al. (1997) that the log P data for highly hydrophobic compounds, especially organochlorines 7 (log P > 6), were underestimated by RP-HPLC. This is the possible ...... 95% confidence intervals reason why many researchers preferred to use software-calculated ------95% predict limits instead of RP-HPLC-determined P values for most of these complex 6 chemicals in the past. However, by using DP-RTC-RP-HPLC, the obtained P shows good consistency with that using SFM/SSM. In contrast, the software-calculated log P has large negative error,

P 5 moreover, which increases with the increase of compound hydrophobicity. For example, the software-calculated log P values are 0 4 5.39 ± 0.36 (with error of 0.30) for p,p -DDD and 6.37 ± 0.37 (with error of 0.59) for p,p0-DDE. Consequently, with the proposed improved DP-RTC method we can expand the application range Literature log 3 of RP-HPLC even for the compounds with log P >6. logK =(1.10± 0.04)logk -(0.60± 0.17) Demare et al. (2007) mentioned that in many works for log P ow w determination by RP-HPLC, the adopted log P reference values of 2 ‘‘model’’ or ‘‘veriﬁcation’’ compounds were often not from SFM/ SSM experiments but software predictions, which could do large 234567adverse effect on measurement accuracy. It can be seen obviously

Logkw from Table 1 that most calculated log P (Clog P) values are smaller than reliable SFM/SSM-measured ones, especially for highly hydro-

Fig. 1. The linear correlation between log P and log kw of model compounds. phobic compounds (log P > 5), which inevitably result in noticeable S.-y. Han et al. / Chemosphere 83 (2011) 131–136 135

Table 3 The validation for DP-RTC-RP-HPLC method.

Compounds Log P (SFM/SSM) a Log P (DP-RTC-RP-HPLC) Error Clog P Error Log P (traditional RP-HPLC) b Naphthalene 3.01 3.06 ± 0.00 0.05 3.45 ± 0.16 0.44 – Biphenyl 3.95 3.80 ± 0.00 0.15 3.98 ± 0.23 0.03 3.63, 3.70, 3.75, 3.93, 4.05, 4.13, 4.26 p,p0-DDD 5.69 5.53 ± 0.02 0.16 5.39 ± 0.36 -0.30 4.73, 4.82, 5.00, 5.19 p,p0-DDE 6.96 6.87 ± 0.02 0.09 6.37 ± 0.37 -0.59 5.63, 5.69, 5.78, 5.83,5.89 6.09, 6.29

a See Table 1 for the source of log P values. b Data for biphenyl from Mackay et al. (2006), and those for p,p0-DDD and p,p0-DDE from Shen and Wania (2005). underestimation of log P if these Clog P values are used for model- Minick et al. (1989) indicated that an ideal situation that the ing. Therefore, abuse of Clog P may be one reason of log P underes- slope of log P–log kw regression is 1 could arrive if RP-HPLC simu- timation by many reported RP-HPLC methods. In our study, the P lates partitioning between n-octanol and water completely. As data extracted from literatures for model and verification com- shown in Fig. 2, for compounds with log P < 4, the log P values pounds were all measured by SFM or SSM, which assured the reli- are almost the same for both of experimental measurement and ability of P determination. theoretical calculation, showing acceptable prediction power of theoretical calculation in this range, in spite that both data points 3.3. Determination of log P for sample compounds are away from the ideal line. However, when log P > 4, negative deviations between Clog P and experimental measured values are distinct, reminding us that theoretical calculation should be dis- Since the correlation between log P and log kw obtained in this work has been validated reliable to predict P of DDT-related com- creetly accepted for highly hydrophobic DDT-related compounds pounds, log P of o,p0-DDT, o,p0-DDD, o,p0-DDE, p,p0-DBP and dicofol in addition to their deviation from ideal line. In contrast, the pro- were determined using DP-RTC-RP-HPLC method and compared posed log P–log kw regression based on DP-RTC-RP-HPLC shows with Clog P (Table 4). The results suggest that the software-calcu- excellent consistency with ideal situation in the range of log P 4– lated data have comparable precision level to the RP-HPLC-deter- 7, which means that this method is especially fit to determine log P mined ones for o,p0-DDD, and have acceptable deviations for values of POPs with strong hydrophobicity. o,p0-DDE and dicofol, but have larger deviations for o,p0-DDT and 0 p,p -DBP. The disparity between software-calculated and RP- 4. Conclusions HPLC-determined log P may be a reflection that the complicated factors such as solute aggregation, solvation or complex functional The lack of reliable experimental P data is a serious problem to groups of DDT-related compounds seriously affect the predictive encumber POPs-related research. It is generally considered that the capability of theoretical calculation in comparison with more key reasons are the strong hydrophobicity and/or high structure simple hydrocarbons. complexity of POPs. RP-HPLC is an important method to determine P, however, the aging of silica-based stationary phase causes the Table 4 change in tR of analyte, especially for high hydrophobic compound Determination of log P for sample compounds. with strong retention, under the same chromatographic conditions Compounds Log P (RP-HPLC-determined) Clog P Error during the measuring course, making uncertain P obtained. In this o,p0-DDT 6.73 ± 0.03 5.92 ± 0.40 0.81 paper, a novel DP-RTC was proposed to rectify the experimental tR o,p0-DDD 5.44 ± 0.01 5.39 ± 0.36 0.05 to eliminate the determination error of P by RP-HPLC. The valida- o,p0-DDE 5.85 ± 0.01 6.22 ± 0.37 0.37 tion results for four DDT-related compounds were in good agree- 0 p,p -DBP 3.94 ± 0.01 4.62 ± 0.33 0.68 ment with those by SFM or SSM, exhibiting better reliability of Dicofol 6.05 ± 0.02 5.74 ± 0.43 0.31 the DP-RTC-RP-HPLC for P evaluation than theoretical calculation and traditional RP-HPLC without DP-RTC, especially for chemicals with log P in the range of 4–7. Moreover, the reasonable DP-RTC- 7 RP-HPLC results of P for five DDTs and dicofol without reliable measured ideal line calculated SFM/SSM data proved that the proposed method could be an effec- tive means in P determination of highly hydrophobic environmen- 6 tal contaminants like POPs.

P 5 Acknowledgments log

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