Analysis of Anionic Surfactants Using Carbon-Chain Branch-insensitive Column

Introduction

Anionic surfactants are widely used as active ingredients in synthetic detergents and serve as indicators of water contamination from industrial effluents and domestic wastewater. In Japan, these compounds are included among the water-quality parameters regulated under the Water Supply Act. The specified analytical method uses high-performance liquid chromatography (HPLC) with fluorescence detection (Notification No. 261 of the Ministry of Health, Labour and Welfare, 2003, Appendix 24) to determine sodium alkylbenzenesulfonates (ABS) with alkyl chain lengths ranging from C10 to C14. The water-quality standard for the combined concentration of these five components is 0.2 mg/L. Testing requires quantification at one-tenth of this value (0.02 mg/L), with a relative standard deviation of less than 20%.

The separation of ABS is influenced by both alkyl chain length and molecular structure. Conventional C18 columns, which contain octadecyl-bonded stationary phases, separate compounds primarily according to differences in hydrophobicity. These columns can also resolve differences in chain branching, producing multiple peaks for compounds with the same carbon number. In contrast, the Wakopak Wakosil AS-Aqua* column separates ABS by alkyl chain length without resolving individual branching isomers, producing a single peak for each carbon-number group. This simplified elution profile facilitates peak identification and quantification. This application note describes the analysis of anionic surfactants using a Wakopak Wakosil AS-Aqua column coupled with highly sensitive fluorescence detection. Recovery results obtained using solid-phase extraction are also presented to assess the method’s suitability for water-quality testing under Japan’s Water Supply Act.

*Wakopak is a trademark or registered mark of FUJIFILM Wako Pure Chemical Corporation.

Experimental

Instruments

Pump: PU-4180*

Autosampler: AS-4050

Column Oven: CO-4060

Detector: FP-4025

*With option units

LC Conditions

Column: Wakopak Wakosil AS-Aqua (4.6 mm I.D. x 250 mm L, 5 µm)

Eluent: 0.1 mM sodium perchlorate in acetonitrile/ultrapure water (65/35)

Flow rate: 0.7 mL/min

Column temperature: 40ºC

Injection volume: 2 µL

Excitation wavelength: 221 nm, Gain x10

Emission wavelength: 284 nm, Gain x10

Standard samples

Mixed standard solutions of anionic surfactants in methanol (1, 2, 5, 10, 25 mg/mL each)

Fig. 1 Structure of sodium alkylbenzenesulphonate (ABS) with various alkyl chain lengths

Keywords

Anionic surfactant, Sodium alkylbenzenesulfonate, Water quality standards, Wakopak Wakosil AS-Aqua, Solid-phase extraction, SPE, Fluorescence detector

Results

Water samples used for testing are concentrated 250-fold by solid-phase extraction (SPE) before analysis; therefore, the calibration range was selected to reflect the concentrations expected after extraction. Table 1 lists the concentrations of each component corresponding to the water-quality standard and their equivalents after 250-fold concentration. Five-point calibration curves spanning 1 to 25 mg/L per component were prepared using triplicate measurements at each concentration. All components exhibited excellent linearity, with correlation coefficients (r) of ≥ 0.9997.

Table 1. Concentration range for calibration curve calculated from standard values for five anionic surfactant components

		Concentration at sampling [mg/L]	Concentration after 250 times condensation [mg/L]
Standard value	Sum of 5 components	0.2	50
Standard value	1 component	0.04	10
One-tenth of standard value	Sum of 5 components	0.02	5.0
One-tenth of standard value	1 component	0.004	1.0
Calibration range	Sum of 5 components	0.02 - 0.5	5.0 - 125
Calibration range	1 component	0.004 - 0.1	1.0 - 25

Figure 2 shows chromatograms from the third injection of the anionic surfactant standard mixture at each calibration level.

Fig. 2 Chromatograms from the third injection of the anionic surfactant standard mixture at each calibration level

Carryover was evaluated by analyzing methanol blanks immediately after the highest-concentration standard (1.0 mg/L each). The blank chromatograms shown in Figure 3 indicate no carryover at or above the lowest calibration level.

Fig. 3 Blank chromatograms for carryover assessment
(chromatogram for standard mixture with 1.0 mg/L each is overlaid)

Table 2 summarizes the measured concentrations of the calibration standards and their accuracy, expressed as a percentage of the nominal concentration. Accuracy ranged from 85% to 104% across all components and concentration levels, meeting the specified acceptance range of 80% to 120%.

Table 2. Accuracy of measured concentrations relative to theoretical values for anionic surfactant calibration standards

Theoretical concentration [mg/L]	Accuracy [%]*	Accuracy [%]*	Accuracy [%]*	Accuracy [%]*	Accuracy [%]*
	C10	C11	C12	C13	C14
1.0	85	88	92	93	94
2.0	96	97	99	98	98
5.0	99	99	99	99	99
10	104	103	102	102	102
25	99	100	100	100	100

*The average of the quantitative values obtained by repeat measurements (n = 3) is used for the calculation.

Figure 4 shows chromatograms from five replicate injections of a standard representing one-tenth of the water-quality standard after 250-fold concentration.

Fig. 4 Chromatograms from replicate injections of the anionic surfactant standard mixture
(1.0 mg/L per component, n = 6)

Table 3 summarizes the peak area repeatability for each component. Relative standard deviations (RSDs) were below 2.0% for all components, demonstrating good injection repeatability.

Table 3. Peak area repeatability for the anionic surfactant standard mixture (1.0 mg/L each, n = 6)

Injection Number	Peak Area [µV・s]	Peak Area [µV・s]	Peak Area [µV・s]	Peak Area [µV・s]	Peak Area [µV・s]
	C10	C11	C12	C13	C14
1	95,636	95,430	91,092	84,322	84,364
2	97,084	93,165	92,978	84,724	80,605
3	95,448	95,579	95,106	86,802	83,755
4	95,513	94,315	89,981	87,910	81,071
5	95,766	92,896	91,402	85,843	83,792
6	95,720	95,890	92,588	85,685	82,991
Average	95,861	94,546	92,191	85,881	82,763
SD	611	1,291	1,788	1,325	1,560
RSD [%]	0.64	1.37	1.94	1.54	1.89

Figure 5 outlines the SPE procedure used to concentrate water samples 250-fold. The specified extraction medium in the Water Supply Act is an octadecyl-bonded silica gel or a material with equivalent or superior performance. An InertSep* C18-ENV SPE column was used in this application note.

* InertSep is a trademark or registered mark of GL Sciences Inc.

Fig. 5 Solid-phase extraction procedure for 250-fold concentration of water samples

Tap water was spiked with the five anionic surfactants at 0.004 mg/L per component and processed according to the procedure shown in Figure 5. Figure 6 presents the resulting chromatograms.

Fig. 6 Chromatograms from the recovery test of tap water spiked with five anionic surfactants and processed by SPE

Table 4 summarizes the recovery results of Figure 6. Recoveries exceeded 85% for all components and were within the specified acceptance range of 70% to 130%, demonstrating satisfactory recovery required by the Water Supply Act from the tap-water matrix.

Table 4. Results of five anionic surfactants from spiked tap water following SPE

Sample	Recovery Rate [%]	Recovery Rate [%]	Recovery Rate [%]	Recovery Rate [%]	Recovery Rate [%]
	C10	C11	C12	C13	C14
Tap water	-	-	-	-	-
Tap water with 0.004 mg/L added	85	89	94	86	87

Conclusion

Measurements of anionic surfactants using a carbon-chain branch-insensitive column showed that the measurement precision could be maintained at ≤20 % at concentrations of one-tenth of the target concentration specified in the Water Supply Act’s water quality standards. A calibration curve was produced by measuring five concentrations of 1.0, 2.0, 5.0, 10 and 25 mg/L, assuming a 250-fold concentration increase after solid-phase extraction, and it was confirmed that the method ensures good linearity and validity.

Recovery tests using tap water spiked at 0.004 mg/L per component yielded recoveries above 85% for all five anionic surfactants, within the specified acceptance range of 70% to 130%. These results demonstrate the method’s suitability for determining anionic surfactants in tap water at concentrations relevant to Japan’s Water Supply Act.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.