DETERMINATION OF ADSORPTION COEFFICIENT OF 

ON SOIL USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

OECD Proposal for a New Guideline 121 

Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge 

Using High Performance Liquid Chromatography (HPLC) 
- 2 - 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality is made for any information contained in this study on the basis of its 

falling within the scope of FIFRA section 10(d)(1)(A), (B) or (C). 

 Company : 

 

 

 
Wildlife International, Ltd. Project Number 523~-112 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: 

This study was conducted in compliance with OECD Principles of Good Laboratory Practice, 

(ENVhlCICHEM (98)17) and Title 40 of the Code of Federal Regulations, Parts 160 and 792, Good 

Laboratory Practice Standards, 1989, with the following exception: 

Characterization and stability of the test substance under storage conditions at the test site were not 

determined in accordance with Good Laboratory Practice Standards. 

Chemist / 

SPONSOR APPROVAL: 

Sponsor's Representative DATE 
QUALITY ASSURANCE STATEMENT 

This study was examined for compliance with OECD Principles of Good Laboratory Practice, 

(ENVMCICHEM (98)17) and Title 40 of the Code of Federal Regulations, Parts 160 and 792, Good 

Laboratory Practice Standards, 1989. The dates of all inspections and audits and the dates that any 

findings were reported to the Study Director and Laboratory Management were as follows: 

DATE REPORTED TO: 

ACTIVITY: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: 

Protocol June 7,2005 June 7,2005 June 22,2005 

Test Substance Preparation July 15,2005 July 15,2005 July 18,2005 

Raw Data and Draft Report September 6-7,2005 September 7,2005 September 13,2005 

Final Report September 14,2005 September 14,2005 September 14,2005 

All inspections were study-based unless otherwise noted. 

hes H. Coleman 

Quality Assurance Representative 

DATE 
-5 - 

REPORT APPROVAL 

SPONSOR: 
TABLE OF CONTENTS 

Title/Cover Page ..................................................................................................................................... 1 

Statement of No Data Confidentiality Claims......................................................................................... 2 

Good Laboratory Practice Compliance Statement .................................................................................. 3 

Quality Assurance Statement.................................................................................................................. 4 

Report Approval ..................................................................................................................................... 5 

Table of Contents.................................................................................................................................... 6 

Summary................................................................................................................................................. 8 

Introduction ............................................................................................................................................ 9 

Purpose ................................................................................................................................................... 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods.......................................................................................................................... 10 

Test Substance.............................................................................................................................. 10 

Reference Standards ..................................................................................................................... 10 

Solvents and Reagents.................................................................................................................. 11 

Mobile Phase Preparation............................................................................................................. 12 

Calibration Stocks and Standards Preparation .............................................................................. 12 

Test Substance Stock and Solution Preparation............................................................................ 12 

Test Procedure.............................................................................................................................. 12 

Data Analysis................................................................................................................................ 13 

Results .................................................................................................................................................. 13 

Conclusions .......................................................................................................................................... 14 

References ............................................................................................................................................ 15 
- 7 - 

TABLE OF CONTENTS 

- Continued - 

TABLES 

Table 1 - Preparation of Reference Stocks and Standard Solutions..................................................... 16 

Table 2 - Typical HPLC Operational Parameters ................................................................................ 17 

Table 3 - Reference Standards Retention Times, Capacity Factors, and Log Koc Values .................... 18 

Table 4 - Adsorption Coefficients (Log Koc) for ................................................................. 19 

FIGURES 

Figure 1 - Reference standard calibration curve .................................................................................. 20 

Figure 2 - A representative chromatogram of a thiourea reference standard ....................................... 21 

Figure 3 - A representative chromatogram of an acetanilide reference standard ................................. 22 

Figure 4 - A representative chromatogram of a phenol reference standard ......................................... 23 

Figure 5 - A representative chromatogram of an aniline reference standard ....................................... 24 

Figure 6 - A representative chromatogram of a naphthalene reference standard................................. 25 

Figure 7 - A representative chromatogram of a phenanthrene reference standard............................... 26 

Figure 8 - A representative chromatogram of a DDT reference standard............................................ 27 

Figure 9 - A representative chromatogram of the test substance, ................... 28 

APPENDICES 

Appendix 1 - Protocol and Protocol Amendment ........................................................................... 29 

Appendix 2 - Example Calculation................................................................................................. 40 

Appendix 3 - Personnel Involved in the Study................................................................................ 41 
- 8 - 

SUMMARY 

Summary: Under the chromatographic conditions specified in guidelines, the test substance eluted as six 

resolved peaks. The corresponding adsorption coefficients (Log Koc) for the test substance 

were calculated as 1.33, 2.43, 3.83, 4.29, 4.34 and 4.61. 
INTRODUCTION

Wildlife International, Ltd. determined the adsorption coefficient of the test substance using the high 

performance liquid chromatography estimation method. The study was conducted at the Wildlife 

International, Ltd. analytical chemistry facility in Easton, Maryland. The study was performed following 

procedures in the OECD Proposal for a New Guideline 121 (adopted January 22, 2001): Estimation of the 

Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid 

Chromatography(HPLC) (1). 

The adsorption coefficient of the test subtance was determined using high performance liquid 

chromatography (HPLC) with ultraviolet detection (UV). The study was identified as Project Number 

523C-112. All raw data generated by Wildlife International, Ltd. and a copy of the final report are filed 

under Project Number 523C-112 in archives located on the Wildlife International, Ltd. site. 

PURPOSE 

The purpose of this study was to experimentally determine an estimate of adsorption coefficient 

(Koc) of the test substance by a reverse-phase HPLC method. 

EXPERIMENTAL DESIGN 

The study was conducted according to procedures outlined in the corresponding protocol (Appendix 

1). The test substance and a series of reference substances bracketing the applicable Log Koc range of the 

HPLC estimation method were separately prepared in either acetonitrile or methanol and diluted in an 55% 

methanol:45% HPLC grade water (v/v) HPLC mobile phase. The reference and test substance solutions 

were sequentially injected into an HPLC system operating under standardized isocratic, reverse-phase 

operating conditions employing a CN-based stationary phase. Capacity factors were calculated for the 

reference standards and test substance using thiourea to estimate column dead time (i.e. the retention time 

of an unretained reference standard). The logarithms of the capacity factors were then plotted against 

published Log Koc values to establish a linear regression calibration. The capacity factors of the test 

substance were used in conjunction with the linear regression equation to calculate the Log Koc range for 

the test substance. 
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MATERIALS AND METHODS 

Test Substance 

The test substance was received from and was assigned 

 

 
- 11 - 

Aniline was received from ChemService on August 12, 2004, and was assigned 

 identification number 6782. The reference standard was identified on the label as 

Aniline; lot/batch number 320-36A; CAS number 62-53-3. The reference standard was a clear liquid, had 

a reported purity of 99.5%, with an expiration date of April, 2009, and was stored under ambient 

conditions. 

stored under ambient conditions. 
Mobile Phase Preparation 

The HPLC mobile phase was 55% methanol: 45% HPLC grade water (v/v). The mobile phase was 

degassed prior to use. 

Calibration Stocks and Standards Preparation 

Seven reference standards were used to generate a Log Koc calibration curve. Calibration stock 

solutions of the individual reference standards were prepared separately by dissolving each in acetonitrile 

or methanol. Calibration standard solutions were prepared from these stock solutions of the reference 

standards by diluting appropriately in 55% methanol: 45% HPLC grade water (v/v). The concentrations of 

the reference standards were adjusted, as necessary, to give a detector response of at least 270 milliabsorbance units for peak height. Details on the preparation of the calibration standards are provided in 

Table 1. 

Test Substance Stock and Solution Preparation 

A stock solution of as prepared by accurately weighing 0.500 g of the test substance into a 

20-mL glass scintillation vial. The test substance was then quantitatively transferred into a 100-mL 

volumetric flask. The stock solution was brought to volume using acetonitrile. This primary stock 

solution contained 5.00 mg/mL. Subsequent dilution of the acetonitrile stock solution yielded a 500 μg 

/mL solution that was prepared using mobile phase. Each of the triplicate injections (100 μL) of this 

solution gave a detector response of at least 100 milli-absorbance units for the peak height of the most 

sensitive peak. 

Test Procedure 

Retention times of the test substance chromatographic components and reference standards were 

determined using an Agilent Series 1100 High Performance Liquid Chromatograph (HPLC) equipped with 

an 

Agilent Series 100 variable wavelength UV detector. Chromatographic separations were achieved using a 

Zorbax CN analytical column (150 mm x 4.6 mm, 5-μm particle size). The column temperature was 

maintained at 40°C. The flow rate was 1.0 mL/minute. Prior to the test substance analyses, the dead time 

was determined by injecting thiourea. The instrument parameters are summarized in Table 2. 
- 13 - 

Reference standards (100 μL injection volume) were injected individually prior to and after the test 

substance to determine retention times. The test substance was injected, in triplicate, following the initial 

reference standard injections and was analyzed under the same chromatographic conditions. 

Data Analysis 

The capacity factor (k′), was calculated for the each of the test substance chromatographic 

components and each reference standard using the following equation: 

k′ = (tR – t0)/t0

where tR was the retention time of the test substance or reference standard and t0 was the column dead time 

established with thiourea. A correlation graph of Log k′ versus Log Koc for the reference standards was 

plotted and fitted to a regression equation in the form y = mx + b. The calibration curve is presented in 

Figure 1. Log Koc for the test substance was calculated by substituting the calculated logarithm of the 

capacity factor for the test substance into the linear regression equation for the calibration curve. An 

example calculation is presented in Appendix 2. 

RESULTS 

Along with analysis of reference standards, a standard of thiourea was injected in duplicate (once 

near the beginning and once near the end of the HPLC sequence) to determine the analytical column dead 

time (t0) for use in calculating capacity factors (k′) of the reference standards and the test substance. The 

mean retention time of the duplicate thiourea standard injections was 2.034 minutes. 

Six additional reference standards were analyzed with the test substance. The reference standard 

series was injected at the beginning and at the end of the HPLC sequence. The capacity factors of each 

standard were calculated based upon their retention times. Calculated Log k′ values for acetanilide, 

phenol, aniline, naphthalene, phenanthrene, and DDT reference standards are presented in Table 3. The 

reference standard calibration curve is presented in Figure 1. Typical reference standard chromatograms 

are presented in Figures 2 through 8. 

The test substance was sequentially injected in triplicate. The test substance eluted as six resolved 

peaks. The mean peak retention times for the test substance ranged from 2.810 ± 0.0993 minutes to 
- 14 - 

11.787 ± 0.568 minutes. The capacity factors (k′) for the minimum and maximum retained test substance 

components were then calculated based on the above retention time range. The corresponding adsorption 

coefficients (Log Koc) for the test substance were calculated to range from 1.33 to 4.61. The calculated 

mean retention times, capacity factors, and mean adsorption coefficients for the test substance are 

presented in Table 4. A representative chromatogram of the test substance is presented in Figure 9. 

CONCLUSIONS 

Under the chromatographic conditions specified in guidelines, the test substance eluted as six 

resolved peaks. The corresponding adsorption coefficients (Log Koc) for the test substance were calculated 

as 1.33, 2.43, 3.83, 4.29, 4.34 and 4.61. 
- 15 - 

REFERENCES

1. Organisation of Economic Cooperation and Development. 2001. Guideline for the Testing of 

Chemicals, 121: Estimation of Adsorption Coefficient (Koc) on Soil and Sewage Sludge using 

High Performance Liquid Chromatography (HPLC). 

2. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. 

D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01: 45-47. 
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Table 1 

Preparation of Reference Stocks and Standard Solutions 

Reference 

Substance 

Amount 

Weighed 

(mg) 

Final 

Volume 

(mL)1

Stock 

Concentration 

(mg/mL) 

Initial 

Volume 

(mL)2

Final 

Volume 

(mL)3

Standard 

Concentration 

(μg/mL) 

Thiourea 500 100 5.00 0.0500 10.0 25.0 

Acetanilide 500 100 5.00 0.0500 10.0 25.0 

Phenol 500 100 5.00 0.200 10.0 100 

Aniline 500 100 5.00 0.0500 10.0 25.0 

Naphthalene 492 100 4.92 0.0500 10.0 24.6 

Phenanthrene 500 100 5.00 0.0500 10.0 25.0 

DDT 500 100 5.00 0.100 10.0 50.0 

1

 Solutions were brought to final volume with acetonitrile or methanol in volumetric flasks. 

2

 Initial volumes measured with a gas-tight syringe. 

3

 Solutions were brought to final volume with 55% methanol:45% HPLC grade water (v/v) HPLC 

mobile phase in volumetric flasks. 
- 17 - 

Table 2 

Typical HPLC Operational Parameters 

INSTRUMENT: Agilent Series 1100 High Performance Liquid Chromatograph 

(HPLC) with an Agilent Series 1100 Variable Wavelength (UV) 

Detector 

ANALYTICAL COLUMN: Zorbax CN (150 mm x 4.6 mm, 5-μm particle size) 

STOP TIME: 30.0 minutes 

FLOW RATE: 1.0 mL/minute 

OVEN TEMPERATURE: 40°C 

MOBILE PHASE: 
Table 3 

Reference Standards Retention Times, Capacity Factors, and Log Koc Values 

Reference 

Standard 

Concentration 

(μg/mL) 

Retention 

Time 

(minutes) 

Capacity 

Factor 

(k′) 1

Log k′

2 Log Koc 3

 

 

 

 

 

 

 

y = 0.3360x -0.8683

R 2 = 0.9779

-1.0

-0.5

0.0

0.5

1.0

1.5

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Log Koc

Figure 1. Reference standard calibration curve. 
- 21 - 

0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\002-0201.D) 2.038

Figure 2. A representative chromatogram of a thiourea reference standard. 
- 22 - 

0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\003-0301.D) 2.623

Figure 3. A representative chromatogram of an acetanilide reference standard. 
0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\004-0401.D) 2.724

Figure 4. A representative chromatogram of a phenol reference standard. 
- 24 - 

0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\005-1501.D) 3.913

Figure 5. A representative chromatogram of an aniline reference standard. 
0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\006-0601.D) 5.025

Figure 6. A representative chromatogram of a naphthalene reference standard. 
0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\007-0701.D) 8.855

Figure 7. A representative chromatogram of a phenanthrene reference standard. 
0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\008-0801.D)

 20.595

Figure 8. A representative chromatogram of a DDT reference standard. 
0 5 10 15 20 25 min

mAU 

0

500

1000

1500

2000

2500

3000

 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\009-0901.D) 2.905 4.017 7.717 10.049 10.363 12.347

Figure 9. A representative chromatogram of the test substance, 
Appendix 1 

Protocol and Protocol Amendment 




- 34 - 
- 35 - 
- 36 - 
- 37 - 
- 38 - 

Appendix 2 

Example Calculation 

Mean dead volume retention time (thiourea) = 2.034 minutes. 

The capacity factor (k′) for the 12.347 minute peak was calculated as: 

k′ = (tR – t0)/t0

where tR was the retention time of the ending peak for the test substance and t0 was the 

column dead time established with thiourea. 

k′ = (12.347 – 2.034)/2.034 

= 5.070 

The logarithm (base 10) of the capacity factor = Log k′ = 0.705 

The n-octanol/water partition coefficient (Log KOW) corresponding to the above capacity factor was 

calculated from the linear regression equation established for the reference standards under the same 

chromatographic conditions: 

Regression equation: y = mx + b, where 

y = Log k′

x = Log Koc

m = slope = 0.3360 

b = y-intercept = -0.8683 

0.705 = (0.3360 × Log Koc) – 0.8683 

 Log Koc = (0.705 + 0.8683) ÷ 0.3360 

Log Koc = 4.68 
- 41 - 

Appendix 3 

Personnel Involved in the Study 

m

 1. Willard B. Nixon, Ph.D., Director of Chemistry 

2. Frank J. Lezotte, B.S., Chemist