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Reference: PMN P-12-0425 

Submission Report NumberPlease find attached the following final report on the PMN substance: 

Determination of Partition Coefficient (n-octanolfwater) of 

Liquid Chromatography (HPLC) Method 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

One confidential copy and one public copy are attached. 

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SANITIZED SUBMISSION 
FINAL REPORT 

Detennination of Partition Coefficient (n-octanoVwater) of 

High Performance Liquid Chromatography (HPLC) Method 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

Submitted to: 

Prepared by: 

May 10, 2013 

1 , . . ·.· 
·· .. :· 

COMPLIANCE WITH THE GLP STANDARDS 

Sponsor 

Study Title Determination of Partition Coefficient (n-octanol/water) of 

High Performance Liquid Chromatography (HPLC) Method 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

Study No. 

i' hereby certify that this study was carried out in accordance with the protocol and 

with our standard operating procedures, and that the report accurately describes the fest 

results. 

The study described in this report was conducted in compliance with the following 

Good Laboratory Practice (GLP) standards: 

OECD Principles of Good Laboratory Practice (1997) 

Study Director 

Sealed date: May 10, 2013 

2 
QUALITY ASSURANCE STATEMENT 

Sponsor 

Study Title Detenninat.ion of Partition Coefficient (n-octanol/water) of 

High Performance Liquid Chromatography (HPLC) Method 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

Study No. 

I verify that this study was carried out in accordance with the following Good 

Laboratory Practice(GLP) and the reported results reflect the raw data of the study: 

OECD Principles of Good Laboratory Practice (1997) 

The types of inspections and their dates, and the dates the results were reported to 

the test facility management and the study director are listed below: 

Protocol: 

Phase of Study 

Inspected 

Draft protocol 

Protocol 

Amendment 01 

Amendment 02 

Amendment 03 

Amendment 04 

In-progress study: 

Date of 

Inspections 

August 7, 2012 

October 16, 2012 

October 29, 2012 

November 26, 2012 

April 10, 2013 

April 12, 2013 

Dates of Reporting to 

Test Facility Management 

and Study Director 

August 9, 2012 

October 16, 2012 

October 30, 2012 

November 26, 2012 

April 10, 2013 

April 15, 2013 

Preparation of test substance solutions October 19, 2012 October 19, 2012 

Measurement of retention time of 

test substance 

Final report: 

Draft report 

Final report 

Quality Assurance Personnel 

October 19, 2012 

November 1, 2012 

May 10, 2013 

October 19, 2012 

November 1, 2012 

May 10, 2013 

Sealed date: May 10, 2013 

3 
{ 

\::. :: 

INTRODUCTION 

1 STUDY TITLE 

Determination of Partition Coefficient (n-octanoVwater) of High Performance 

Liquid Chromatography (HPLC) Method, OECD Guidelines for the Testing of Chemicals 117 (2004) 

2 PURPOSE 

This study was conducted to determine the partition coefficient (n-octanoVwater) of the 

test substance. 

3 TEST GUIDELINE 

OECD Guidelines for the Testing of Chemicals 117 (2004) "Partition Coefficient Znoctanol/water), High Performance Liquid Chromatography (HPLC) Method" 

4 GLP COMPLIANCE 

OECD Principles of Good Laboratory Practice (1997) 

5 SPONSOR 

6 TESTING FACILITY (Head Office) 

7 TESTING FACILITY (Laboratory) 

8 STUDY DIRECTOR 

4 
( 

9 EXPERIMENTAL SCIENTIST 

(Experimental work) 

10 STUDY PERIOD 

Study initiation: 

Experiment: 

Study completion: 

October 15, 2012 

October 19, 2012 

May 10, 2013 

11 STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS 

The followings will be stored in the archives of the testing laboratory. 

1) Protocol 

2) Final report 

3) Raw data 

4) Test substance 

5) Reference substance 

6) Other requirements 

5 
( 

CONTENTS 

Page 

SUMMARY···· ·················· · ··················································· · ··7 

TEST SUBSTANCES· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 

1.1 Name, structural formula and physico-chemical properties· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 

1.2 Source········································· ·· ·· · ··························· 10 

1.3 Stability under the storage conditions·· · · · · · · · · · · · · · · · · · ·: · · · · · · · · · · · · · · · · · · · · · · · · · · 10 

2 TEST METHOD · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11 

2.1 

2.2 

2.3 

2.4 

2.5 

2.6 

2.7 

2.8 

Reference substances · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11 

Reagents · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 12 

Apparatus··········· · ··· · ·· · ······· · · · ····· ·· ····· ··········· · · ······ · ······· · · 12 

Analytical method · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 12 

Preliminary estimate of the partition coefficient · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 12 

Preparation of reference substance solutions · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 13 

Preparation of test substance solutions· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · }).3 

Verification of correlation between partition coefficient and retention time · · · · · · · · · · · · · · · 14 

2.9 Measurement of retention time and calculation of partition coefficient · · · · · · · · · · · · · · · · · · · 14 

2.10 Validity of the test·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 15 

3 MEASUREMENT of pH of The WATER EXTRACT of The TEST SUBSTANCE -1 · · · · · · 15 

3.1 Measurement of pH · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 15 

3 .2 Apparatus · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 15 

4 MEASUREMENT of pH of The WATER EXTRACT of The TEST SUBSTANCE -2 · · · · · · 15 

4.1 Guideline · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 15 

4.2 Measurement· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 16 

4.3 Apparatus and reagent · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 16 

5 RESULTS AND DISCUSSION··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 16 

5.1 Deviation and circumstance that might have affected the reliability of the test result · · · · · · 16 

5.2 Results of the measurement of the Jog Pow · · · · · · · ······ ····· ················· · · · · · · 16 

5.3 Results of the measurement of the pH · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 17 

Table 1 Results of determination · · · · · · · · · · · · · · · · · · · · · · ·: · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 18 

Figure l Correlation diagram between log Pow and log k · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 19 

Figure 2 ~omatograms · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · : · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 20 

Appendix 1 Infrared absorption spectra .................................................................................................... 22~23 

Appendix 2 High performance liquid chromatograph (HPLC) analytical conditions ................................ 24~25 

6 
SUMMARY 

Sponsor: 

Study title: 

Determination of Partition Coefficient (n-octanol/water) of 

High Performance Liquid Chromatography (HPLC) Method 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

Study number: 

Test method: 

OECD Guidelines for the Testing of Chemicals 117 (2004) 

''Partition Coefficient (n-octanol/water), 

High Performance Liquid Chromatography (HPLC) Method" 

1) Reference substances : Methyl Benzoate 

Ethyl Benzoate 

1,4-Dichlorobenzene 

Cumene (lsopropylben7...ene) 

1 ,2,4-Trichlorobenzene 

Triphenylamine 

2) Analytical method: High Performance Liquid Chromatograph (HPLC) 

7 
( 

Results: 

The peaks of the test substance were detected on the chromatograms, Based on order of the 

retention time, component A-F were defined. The partition coefficient (log Pow) value of 

each component of the test substance was calculated and was shown below. Component A 

was presumed to be one of the impurities in the test substance. Because the retention time of 

Component A was very different from the retention time of the major components in the · 

test substance, the structures of the major active ingredients in the test substance are very 

similar and suggests that log Pow for these components should be similar, the log Pow for 

components was very different from the log Pow for the major components in the test substance and also very different from the predicted log Pow( 4.87) . 

Component log Pow 

A 2.8 

B 4.5 

c 4.6 

D 4.6 

E 4.8 

F 4.9 

8 

I 

I 
1 TESTSUBSTANCES 

1.1 Name, structural formula and physico-chemical properties 

Nameofthe I 

chemical substance I 

Alternate name i 

CASRN 

I 

I 

l 

Structural formula or I 

rational formula I 

(ifboth ofthem are ! 

unknown, outline of i 

~ preparation or ! 

manufacturing) 

I 

Molecular weight 362.11 

Purity{%) of the 

chemical substance 99.47 

subjected to the study 

Lot number of the 

chemical substance 

subjected to the study 

Name and content of 

impurities 

Vapor pressure 2.9kPa (25CC) 

Solubility in water ~1001 mg/L 

1-octanol/water partition 

coefficient -

Melting point -

Boiling point llOCC(Representative value) 

Appearance Liquid at room temperature 

Stability Stable 

9 
( . 

Solubility in Solvent Solubility Stability in solvent 

solvents and the like Acetone ;;;;87889 mg/L -

Above mformatlon IS provtded by the sponsor. 

1.2 Source 

Supplier: 

1.3 Stability under the storage conditions 

During the study, the test substance was stored in a desiccator in the testing laboratory 

(storage condition: room temperature, dark, air-tight). 

· After the completion of the experiment, the infrared absorption spectrum of the test 

substance was measured. Stability of the test substance was confirmed by comparing the 

spectrum with that measured before the start of the experiment. 

[Appendix 1] 

Apparatus: Infrared spectrophotometer Nicolet iS 10, Thermo Fisher Scientific Inc. 

10 
( 

~. 

2 TEST METHOD 

The study was conducted in accordance with the OECD Guidelines for the Testing of 

Chemicals 117 (2004) "Partition Coefficient (n-octanol/water), High Performance Liquid 

Chromatography (HPLC) Method''. 

Six substances with known partition coefficients (see 2.1) were selected as reference 

substances by preliminary examination carried out before the study initiation. Thiourea (see 

2.2) was selected as a non-retainable substance, for determination of the dead time (fc). 

2.1 Reference substances 

No. Name CASRN Structural formula 

Supplier J Reagent Grade Lot No. Purity 

1 Methyl benzoate 93-58-3 Hl\ 

o-< ~ 

Wako Pure Chemical ! 

Industries, l.td., [Wako guaranteed grade KWH4529 99.9% I 

2 Ethyl benzoate 93-89-0 / H3 

Wako Pure Chemical ! o-<-CR, Industries, Ltd., !Wako guaranteed grade KWP4133 99.9% 

3 I ,4-DicWorobenzene 106-46-7 Cl--o--CI 

Tokyo Chemical Tokyo Chemical 

ICBHIB 99.9% Industry Co., Ltd. guaranteed grade 

4 Cumene (Isopropylbenzene) 98-82-8 o-r· 

Tokyo Chemical I Tokyo Chemical c\ J 6YAKC 99.9% CH1 

Industry Co., Ltd. !guaranteed grade I 

I 

I Cl 

5 I ,2,4-Tricl\lorobenzene 1120-82-1 

Tokyo Chemical I Tokyo Chemical ftrst 

I 

3LYYC 99.6% 

~ # a 

[ndustry Co., Ltd. grade Cl I 

6 Triphenylamine I 9 603-34-9 

Kanto Chemical Co., I 

Inc. guaranteed grade 004W2038 100.0% UND 

11 
L 

2.2 Reagents 

2.3 

2.4 

Thiourea: Guaranteed grade, Kanto Chemical Co., Inc. 

Purity 98.9%, Lot No. 107N2156 

Methanol: HPLC grade, Wa.ko Pure Chemical Industries, Ltd. 

Sodium Dihydrogenphosphate Dihydrate: Guaranteed grade, 

Wa.ko Pure Chemical Industries, Ltd., 

Disodium Hydrogenphosphate 12-Water: Guaranteed grade, 

Wa.ko Pure Chemical Industries, Ltd., 

Ultra pure water: Grade A4 in Japanese Industrial Standards (JIS) K0557 

Apparatus 

Electro balance: AG204, Mettler K.K. 

PB3002, Mettler K.K. 

pH meter: Twin pH B-212, Horiba, Ltd. 

Analytical method 

.:: 

The retention times of the test substance and reference substances were measured with 

high performance liquid chromatograph (HPLC). Analytical conditions are shown in 

Appendix 2. The pH of the HPLC eluent (10mM sodium phosphate buffer' :methanol= 

25 : 75 ) was 7.9. 

*Preparation of 1 OmM sodium phosphate buffer : 0.78 g of sodium dihydrogenphosphate and 1.79 g of dihydrate disodium hydrogenphosphate 12-Waterwere dissolved to 1 

L with ultra pure water. 

2.5 Preliminary estimate of the partition coefficient 

The value of the partition coefficient (log Pow) ofthe test substance was estimated to be 

4.87, by the testing laboratory using the EPI (Estimation Programs Interface) Suite ver. 

4.10. KowWin ver. 1.68 (U.S.EPA, 2011). 

12 
·: .. ·. 

l 

' . \. 

2.6 Preparation of reference substance solutions 

Following the table below, duplicate solutions of the reference substances were prepared by 

mixing appropriate volumes of 1 OmM sodium phosphate buffer, the stock solution of thiourea 

(dissolved in methanol) and the stock solutions of each reference substance (dissolved in 

methanol). The relative proportion of the solvents in the mixed solution was equal to the 

eluent of the HPLC. 

Partition Concentration Volume Concentration in 

Coefficient . of the stock collected the final solution Name solution (log Pow) (mg!L) (f.lL) (mg/L) 

10 mM Sodium phosphate buffer - 375 -

Methanol - - 855 -

Thiourea - 500 30 10 _, 

., 

Methyl benzoate 2.1 1000 40 27 

Ethyl benzoate 2.6 1000 40 27 

1 ,4-Dichlorobenzene 3.4 1000 50 33 

Cumene (Isopropylbenzene) 3.7 1000 50 33 

1 ,2,4-Trichlorobenzene 4.2 2000 20 27 

Triphenylamine 5.7 1000 40 27 

Total volume of Methanol 1125 (J.L 

Total volume of the final solution 1500 f.1L 

Concentration of Methanol 75% 

* : OECD Guidelines for the Testing of Chemicals 117 (2004) 

2. 7 Preparation of test substance solutions 

The 125.8 J.LL (specific gravity: 1.59•) portion of the test substance was dissolved to 20 mL 

with methanol as stock solution. Following the table below, duplicate mixed solutions of the 

test substance were prepared by mixing lOmM sodium phosphate buffer, the stock solution of 

thiourea (dissolved in methanol) and the stock solution of the test substance (dissolved in 

methanol). The relative proportion of the solvents in the mL'(ed solution was equal to the eluent of the HPLC. 

* : measured value obtained by our testing facility 

13 
\ 

( 

Concentration 

Volume Concentration in 

of the stock 

Name collected the final solution 

solution 

(mg/L) (~) (mg/L) 

1 OmM Sodium phosphate buffer - 375 -

Methanol - 845 -

Thiourea 500 30 10 

Test substance 10000 250 1670 

Total volume of Methanol 1125 ~ 

Total volume of the final solution 1500 ~ 

Concentration of Methanol 75% ~ 

2.8 Verification of correlation between partition coefficient and retention time 

The duplicate reference substance solutions were analyzed with HPLC. The retention 

times (min.) of the reference substances and thiourea were determined from the 

chromatograms. The retention time of the reference substances (tR) was corrected by 

subtracting the retention time of thiourea (to), i.e. tR- to. The capacity factor (k) was 

calculated according to the following equation. 

k = (tR- to) I to 

The log Pow for each reference substance was plotted against its logarithmic value of 

capacity factor (log k), and the regression line was drawn by the least squares method. 

log Pow = a + b log k 

where a: y-intercept of regression line 

b: slope of regression line 

A correlation coefficient (r) between log k and log Pow was determined to verify the 

linearity. Criteria for linearity is that r value is greater than or equal to 0.98. 

2.9 Measurement of retention time and calculation of partition coefficient 

The duplicate test solutions of the test substance were analyzed with HPLC. The 

retention times (min.) of the test substance and thiourea were determined from the 

chromatograms. The logarithmic value of the capacity factor (log k) of the test substance 

was calculated from the retention time of the test substance (tR) and that of thiourea (to). 

The Partition coefficient (log Pow) of the test substance was calculated using the linear 

regression formula derived in Section 2.8. 

14 
2.10 Validity ofthe test 

• The value of log Pow determined from duplicate measurements should fall within a range 

of±0.11ogunits. 

• The temperature during the measurement should not vary by more than ±1 °C. 

3 MEASUREMENT of pH of The WATER EXTRACT of The TEST 

SUBSTANCE -1 

The pH of the water extract of the test substance was measured. 

( 3 .1 Measurement of pH 

The 10.0 g 6289~) portion of the test substance and the 10.0 g of ultra pure watJr* 

were entered in the 3 0 mL-separating funnel, mixed with shaking apparatus for 5 min. and 

leaved to separate to two layers completely. Upper water layer was sampled and the pH 

was measured. 

*Ultra pure water: Grade A4 in Japanese Industrial Standards (JIS) K0557 

pH5.3 (measured value) 

3.2 Apparatus 

pH/D.O. meter : MM-60R, OKK-TOA Corporation 

Shaking apparatus : SA-31 Yamato Scientific Co.,Ltd. 

4 MEASUREMENT of pH of The WATER EXTRACT of The TEST 

SUBSTANCE -2 

The pH of the water extract of the test substance was measured in accordance with the 

Guideline "ASlM standard 02110-00 (Reapproved 2012)" to obtaine the pH-value correctly. 

4.1 Guideline 

ASTM standard 02110-00 (Reapproved 2012) 

15 
4.2 Measurement 

1 Preparation of the water 

Approximately 1000 mL of the ultra pure water was sampled in the 1 000-mL Pyrex 

beaker, boiled for 5min with electric hot plate, then covered and cooled to room temperature. A 250 mL aliquot of the water (pH 5.8) was to pH 7.3 with the titration of 0.23 mL of 

0.01 N NaOH. The amount of0.46 mL was added to 500 mL water and confirmed pH of 

7.1. 

2 Pretreatment 

Aliquot of 50 mL of the test substance was transferd to a 200-mL separatory funnel containing 50 mL of Ultra pure water (pH 7.1) prepared by the above procedure, shaken the 

mixture for 30 min, then let stand for 30 min. The water extract was collected and the pH 

was determined with pH meter(n=S). 

4.3 Apparatus and reagent 

pH/D.O. meter : MM-60R, DKK-TOA Corporation 

Shaking apparatus : SA-31 Yamato Scientific Co.,Ltd. 

0.01 moVl Sodium Hydroxide Solution (N/100) : Kanto Chemical Co., Inc. 

5 RESULTS AND DISCUSSION 

5.1 Deviation and circumstance that might have affected the reliability of the test result 

There was no deviation or specific factor that might have affected the reliability of the 

test results. 

5.2 Results of the measurement of the log Pow 

The measurement results are shown in Table 1, correlation diagram between log Pow 

and log k is shown in Figure 1, and HPLC chromatograms are shown in Figure 2. 

The regression line formula is shown below. The correlation coefficient r was fulfilled 

the criteria, r 0.98. 

log Pow = 1.704 + 2.867 x log k 

r = 0.9968 

The peaks of the test substance were detected on the chromatograms, Based on order of 

the retention time, component A-F were defmed. The partition coefficient (log Pow) value 

16 
t.. ... 

of each component of the test substance was calculated and was shown below. This study 

was judged valid since the value of log Pow derived from repeated measurements fell 

within a range of± 0.1 log units, and the temperature during the measurement was within a 

range of± 1 °C. Average values shown below are judged to be log Pow of the test substance. Component A was presumed to be one of the impurities in the test substance: 

~ecause the retention time of Component A was very different from the retention time of 

the major components in the test substance, the structures of the major active ingredients in 

the test substance are very similar and suggests that log Pow for these components should 

be similar, the log Pow for component A was very different from the log Pow for the major 

components in the test substance and also very different from the predicted log Pow(4.87). 

comPOnent Actual measurement Average 

A 2.85 2.8 

2.84 

B 4.49 4.5 

4.49 

c 4.55 4.6 

4.55 

D 4.65 4.6 

4.64 

E 4.79 4.8 

4.79 

F 4.85 4.9 4.85 

5.3 Results of the measurement of the pH 

The pH of the water extract prepared according to the procedure described in Section 

4.2 was measured to be 4.7. The measured values were 4.7, 4.5, 4.7, 4.8 and 4.6(n=5). 

Methoxytridecafluoroheptene which is the main component of the test substance is nondissociative and hardly-soluble in water. Thus, the main component in water extract is 

thought to be very few. So, it was considered that the obtained pH value did not represent 

the property of main component. 

The pH of the water extract prepared according to the procedure described in Section 

3.1 was measured to be 4.5 

17 
Table 1 Results of measurement 

I Peak jchromato1 to or tR I k l logk log Pow 

Name ! gram I I 

i No. No. 1 (min) I i 

Thiourea 

I 

1 I ! 1.440 I - I 

I - - (Dead time measurement to) 2 1.441 - - Methyl benzoate I 2 1 I 3.313 I 1.301 I O.li4 2.1 

I 2 ; 3.312 j 1.298 0.113 I Q) 

Ethyl benzoate I 3 1 . 4287 1.977 I 0.296 2.6 () I 

~ l I 

2 I 

{I) 4.286 1.974 l 0.295 I .0 

::: 1 ,4-Dichlorobenzene I 4 1 i {I) 7.278 4.054 1 o.6o8 3.4 Q) 2 I 7.274 I 4.048 1 o.6o7 () I 

= Q) 

. Cumene (lsopropylbenzene)l 5 1 9.711 5.744 0.759 3.7 t.J 

.a 

OJ'. 2 1 9.7o5 I 5.735 I 0.759 : I 

I ,2,4-Trichlorobenzene l 6 I 1 12.109 . 7.409 0.870 4.2 " 

2 12.100 l 7.397 0.869 I 

Triphenylamine 1 7 1 34.335 j 22.844 I 1.359 5.7 

I 2 1 34.306 I 22.807 1358 

Thiourea I 1 1 1.441 I - I 

! - - (Dead time measurement: to) 2 i ) 1.440 - - ' 

l 2 I I 5.043 2.500 l 0.398 2.85 !Average Component A l 

2 ! 5.035 2.497 1 o.397 2.84 2.8 

I 

3 1 l 14.983 I 9.398 I 0.973 4.49 !Average Component B I 

2 114.943 9.377 I 0.972 4.49 1 4.5 ~ I 

... c I 4 1 1 15.643 9.856 ! 0.994 4.55 tAverage II) tQ Component C Q) -

E-<~ 115.653 I 4.55 ! 4.6 i 2 9.870 i 0.994 ;:I ! II) 

I 5 1 16.743 10.619 I 1.026 4.65 iAverage ComponentD 2 16.699 10.597 I 1.025 4.64 4.6 ! 

I 

i 6 1 118.665 1 11 .953 I 1.077 4.79 Average Component E i I 

2 1 18.616 1 11.928 1.077 4.79 4.8 

I 7 1 19.484 1 12.521 I 1.098 4.85 Average 

ComponentF I ! 

! 2 I9.432 12.494 1.097 4.85 4.9 

to: Dead ttme 

tR: Retention time of reference substance or test substance 

k: Capacity factor 

Equations: k = (tR-to) I to 

Log Pow of the test substance is calculated using the following regression formula 

Regression formula calculated from the reference substances data 

log Pow= 1. 704 + 2.867 x log k 

r= 0.9968 

18 
Figure 1 Correlation diagram between log Pow and log k 

6.0 

5.0 

4.0 

~ 0 

Q.. 3.0 

bO 

0 

- 2.0 

1.0 

0.0 

0.0 

y= 1.704 + 2.867 X 

r= 0.9968 

0.5 

log k 

19 

I ! 

..--- -,1 I 

• Reference substance 

OComponent A 

0 Component B 

!:!:. Component C 

I 0 Component D 

X Component E 

+Component F 

1.0 1.5 
Figure 2 Chromatograms 

Reference substances, No. 1 

··-

100 ! 

~ :; ; ;; 

~ .: :;; eo 

.., 

to 

0 

,;, ... 

t\ ,;, 

·-------..~~··· 

-·--·---·-·--·-··-----·-· 

t"·t lT tff • I fah) (ala) 

1.~0 .. 0. 011 uu !t.S U U ll .. o.m 650. 4 UIT .. o.m 'tS.I f.%11 .. O.Zll llU S. fll • o.w l~f 4 

11 .1 ·-~ 53. f T.O 0.4 11.0 fl.l IS.t lLiot • o.m 1444.5 S4.SJI • o.n1 10tf.4 B . t au 3U 2.9.0 

: UILJ 

---.-----~ • u IDd ------~--------------­ or IQor t ... 

Reference substances, No.2 

""'-V 

i 100 ~ 

! eo .. :! 

~ I! .: "' eo 

I i .., 

A Jt 

0 

... .. .~ 

-~----·illlll"·t>ll«'·l ------------------

1. 441 • O. OM sou t7.0 ?. 1 1.111 Fl o.m 591.7 14.1 '-4 UN 1111 0.151 191.! su f.l t.m Ill o. n1 lit. I IL l 11.1 t. TOI 1111 o. w 101U fU IS. I U .IOO 1111 0.341 1515. I lS.S 11. s U.JOI Ill 0.971 Ull.S n.1 11.0 

)·1•: 1051.1 4tl. l 

._...,.._••..-- •• .. •-•-wmu 

... ___ tu hd of lt»rt '*'* 

20 
( 

"\ ,, 

Figure 2 Chromatograms (continued) 

Test substance, No.1 

J •.AU 

i 140 " 

to 

10 

<10 

20 b Q 

=~ 0 :l:!. fi. ..... 

•• 

t'-t It J!t II • (lila) (liJaJ 

1.441 • s.ou • 1~1*1 • 1$.14) • lf. 7U • lUIS • lt.IM Ill 

Hl: 

O.OM 

o. uo o.no 

o.m 

0.$11 

o.m 

0.411 

i 

:! 

h 

... 

~"' "' 3.0 

1. 4 m.s IIIII.S 

lOQ.G 

l7U.t 

U t. W ofltocrt t .U 

Test substance, No.2 

·~· j~ 

100 

10 

10 

.. 

9T.I 0.5 

0. 1 0.1 

1.1 

45.1 

10. 5 

15l.4 

---

·---~ ------ ·--.;.. ------·--~~~---------­ · ,;;llliii';.;;.;'_·~;,.IW;.;;..·;.~---~= 

t'·t 

' l410 1'1 S.OlS l1 11.111 l1 IU$1 l1 lUll l1 lUll llr lt. 4ll I'll 

Hrr: 

(o~ 

0.011 

0.150 

O.IU 

o.m 

0.531 

0.120 

o. cn 

!U.t 

u 

l.O 

I. I m.a IT!6.1 

IIU 

mu 

110. 4 0.1 

0.1 

0.1 

7.4 

"·I If. I 

115. 1 

11.) 

1. 2 

0.1 

G. I 1.1 

ll.t 10.1 

II. I 

O.l 

0.1 

o.o 

1.5 

11.1 

10.1 

.... ___ ""-·-----~,....~-~ Ut bd of te.orl Ut 

21 
Appendix 1 

Infrared absorption spectra 

22 
Figure A-1-1 Infrared absorption spectrum of the test substance before the measurement 

• 0 

i 

,i 

II 

100 

90 

90 

70 

liO 

50 

40 

30 

20 

10 

4000 

8#: * Jun 29 13:19:40 2012 (GMT009:00} * 

A~:.-:32 

~-- •tOOO 

I 

~~I ... 

;;:; 

2500 2000 1SOO 

Wwe~ cm-1 

Figure A-1-2 Infrared absorption spectrum of the test substance after the measurement 

100 

y ~ T 

~ '~ 90 

I 80 w~. I 

I 10 

60 

~ ~ 8 ,,.,. ... c; 

!! <0 

" so ... .. 

"' .. "" ... ~ 

..... 

40 

I - 30 !::: ... ... 

~ 

20 

10 }e 

I 0 ::! -

I 4000 3500 3000 2500 2000 1500 1000 

w.v......,.,.,.Ccm-1) 

8#: * Apo-1213:08:07 2013(GMT-+0~) • *Apr 12 13.1l8:07 2013 (GMT-+09!10) Ap!W'&tu.S: Nleolet 1510 

;tud• NumMr! - A~'\<z32 "est ubttan~ - l.otN ~~~J~:~~: ·A.-.( IJI"(f>'£--;;;:;- ~-C: 4.000 m 

" L "1. 

23 
High Performance Liquid Chromatograph (HPLC) Analytical Conditions 

System 

High performance liquid chromatograph Agilent 110.0 (No.6), Agilent Technologies 

Work station: 

De gasser: 

Pump: 

Auto sampler: 

Column oven: 

Photodiode array detector (DAD): 

Conditions 

Chemstation 

Gl379A 

G1311A (Quaternary pump) 

Gl313A 

G1316A 

Gl315B 

Column: lnertsil ODS-3, 5 JliD, 3.0 m.m i.d. x 150 mm, GL Sciences Inc. 

Column oven temperature: 40°C 

Eluent: lOmM sodium phosphate buffer : methanol= 25: 75 (pH 7.9) 

Flow rate: 

Wavelength: 

Injection volume: 

0.5 rnUmin 

210nm 

25