Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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Prepared Checked/Approved valid as of

H. Meuth DI M. Hirschen 08.05.09 

Title: Quantitative determination of Decanoyl oxy benzoic acid 

(DOBA) and Hydroxy benzoic acid (HBA) in DOBA samples by 

HPLC 

Contents

1. Objective and scope 

2. Principle 

3. Apparatus 

4. Reagents 

5. Procedure 

6. Validation of the method 

7. Remarks 

8. Further applicable documents 

9. Literature references 

10. Appendix 

Validation: Precision 

 Accuracy 

 ________________________ 

 ________________________ 
Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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1. Objective and scope 

The method is used to determine the content of decanoyl oxy benzoic acid (DOBA) and hydroxy 

benzoic acid (HBA) in DOBA samples by HPLC. 

2. Principle 

The DOBA and HBA content are determined by reversed phase HPLC with UV detection. 

3. Apparatus

HPLC instrument with dual gradient pump, e. g. Agilent 1100 

UV detector, e. g. Agilent 1100 

HPLC column, Synergi Polar RP, 250 mm x 4,6 mm ID, 4 μm (Phenomenex, No. OOG-4336-EO) 

Ultrasonic bath 

Ordinary laboratory equipment 

4. Reagents

4.1 Acetonitrile, HPLC grade, e.g. Riedel de Haen No. 34851 

4.2 2-Propanol, HPLC grade, e. g. Riedel de Haen No. 34863 

4.3 Tetrabutyl ammonium hydrogen sulphate (TBAHS), puriss., e. g. Fluka No. 86868 

4.4 Solvent 1: 

2-propanol / acetonitrile / water = 5 / 3 / 2 (v/v/v) 

4.5 Solvent 2: 

acetonitrile / water = 1 / 1 (v/v) 

4.6 Eluent A: 

water / acetonitrile = 95 / 5 (v/v) + 0,005 mol/l TBAHS 

4.7 Eluent B: 

water / acetonitrile = 5 / 95 (v/v) 

4.8 DOBA reference substance with known DOBA content, e. g. BES 04101, w = 99,9 % (w/w) 

4.9 4-Hydroxybenzoic acid reference substance with known content, e. g. Aldrich No. 240141 
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5. Procedure 

5.1 Sample preparation 

Weigh to the nearest 0,1 mg an appropriate amount of the test substance (e. g. approx. 50 mg dry 

filter cake and approx. 60 mg wet filter cake, respectively) into a 10 ml measuring flask. 

Add approx. 8 ml solvent 1 (4.4) and treat with ultrasound until the substance is completely 

dissolved. 

Let the solution cool to room temperature and fill to the mark with solvent 1 (4.4) 

Dilute sample solution 1:10 with solvent 2 (4.5). 

Inject 10 μl of the final sample solution. 

5.2 Calibration 

5.2.1 DOBA 

Weigh to the nearest 0,1 mg approx. 20 mg, 40 mg and 60 mg DOBA reference substance (4.8) 

each into a 10 ml measuring flask. 

Add approx. 8 ml solvent 1 (4.4) and treat with ultrasound until the substance is completely 

dissolved. 

Let the solutions cool to room temperature and fill to the mark with solvent 1 (4.4) 

Dilute each stock solution 1:10 with solvent 2 (4.5). 

Inject 10 μl of each final calibration solution. 

5.2.2 HBA 

Weigh to the nearest 0,1 mg approx. 35 mg and 65 mg HBA reference substance (4.9) each into a 

100 ml measuring flask. 

Dissolve and fill to the mark with solvent 1 (4.4).

Dilute each stock solution 1:1000, 1:5000 and 1:10000 with solvent 2 (4.5). 

Inject 10 μl of each final calibration solution. 
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5.3 Evaluation 

Calculate all final solutions in mg/l. Evaluation is done by external standard calibration via linear 

regression line. 

m

100

a

A b

w

i

i

⋅

−

=

Where is: 

wi

 content of DOBA and HBA, respectively in % (w/w) 

Ai

 peak area of DOBA and HBA, respecitvely 

a slope of regression line 

b axis intercept of regression line 

m sample weight in final sample solution in mg/l 

5.4 HPLC-Conditions 

Chromatography column/s: 

 Stationary phase length 

[mm] 

ID 

[mm] 

particle

size 

[μm] 

Pre-column: none --- --- --- 

Column: Synergi Polar RP 250 4,6 4 

Mobile Phase: 

A: water / acetonitrile = 95 / 5 (v/v) + 0,005 mol/l TBAHS (4.6) 

B: water / acetonitrile = 5 / 95 (v/v) (4.7) 

C: --- 

Gradient: T [min] % B isocratic: A: --- % 

 0 30 B: --- % 

 5 30 C: --- % 

 15 90 

 20 90 

 22 30 

 30 30 

Run time: 30 min 

Flow rate: 1,0 ml/min 
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Column temperature: 23 °C 

Injection volume: 10 μl 

Detection: 

UV detector Response time: >0,05 s 

DA detector Slit: 4 nm 

 Wavelength/BW: 254 / 4 nm 

 Reference wavelength/BW: 400 / 100 nm 

6. Validation of the method 

Precision

6-fold determination of DOBA content in sample CUS 06-597: 

n1: 99,2 n4: 99,3 

n2: 99,1 n5: 97,7 

n3: 99,7 n6: 99,9 

mean: 99,2 

std. deviation: 0,77 

Coeff. of variation: 0,8 

Precision

6-fold determination of HBA content in sample CUS 06-597: 

n1: 0,07 n4: 0,06 

n2: 0,07 n5: 0,06 

n3: 0,07 n6: 0,07 

mean: 0,07 

std. deviation: 0,005 

Coeff. of variation: 7,7 
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Linearity of DOBA calibration:

Conc. [mg/l] area [u] Slope: 6,3936 u/(mg/l) 

202,797 1393,35541 Intercept: 102,9990 u 

415,584 2772,73975 Correlation coeff.: 0,9999 

622,377 4075,79541 Residual std. deviation: 15,5162 u 

 Method std. deviation: 2,4268 u/(mg/l) 

 Rel. method std. dev.: 0,59 % 

Linearity of HBA calibration:

Conc. [mg/l] area [u] Slope: 59,3891 u/(mg/l) 

0,071 7,20713 Intercept: 2,8871 u 

0,122 10,17547 Correlation coeff.: 0,9998 

0,357 23,76888 Residual std. deviation: 0,2486 u 

0,608 39,17189 Method std. deviation: 0,0042 u/(mg/l) 

 Rel. method std. dev.: 1,45 % 

7. Remarks

For the determination of by-products, e. g. C8- and C9-OBA a DOBA calibration is used since no 

reference materials exist. For the calibration of the by-products determination 20 mg, 40 mg and 

60 mg DOBA reference substance are each dissolved in 100 ml solvent 1 (4.4) and diluted 1/10, 

1/100 and 1/1000 with solvent 2 (4.5) in the same way as described for the DOBA calibration. 

8. Further applicable documents

none 

9. Literature references

none 
Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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10. Appendix

Typical Chromatogram: