**3. Separation, identification and quantification of synthetic surfactants**

Over the last decades, analysis of surfactants in environmental samples has been carried out using several instrumental techniques. So far, spectrophotometric, potentiometric titrametration (PT) and tensammetric tecniques have been optimized to measure the total content of ionic [112-114] and non-ionic surfactants [115, 116], although their sensitivity and/or specifity tend to be low compared to chromatographic techniques coupled to several types of detectors. Generally, one of the main applications of spectrophotometric techniques has been routine environmental analysis due to their quickness and simplicity. They involve the formation of ion associates of analytes with specific ion-pair reagents and their extraction into appropriated organic solvents. After phase separation, the absorbance of the organic phase is measured. However, despite the advantages described above, the use of spectrophotometry generates very toxic wastes (e.g., chloroform) and is only limited to the analysis of total amount of surfactants [117-119]. PT and tensammetric techniques [120, 121] are based on the changes in electric properties caused by the presence of analytes in environmental samples. They can be only applied to the determination of total ionic and nonionic compounds, being impossible for both techniques to discriminate among individual components from surfactant mixtures. Besides, there are also issues associated with reproducibility and signal stability [113]. Nowadays, it is necessary to go beyond quantification of the total concentration of target analytes and, in most cases, chromatographic techniques (gas chromatography, GC, or high-performance liquid chromatography, HPLC) coupled to various types of detectors are preferred to separate and identify each individual compound from surfactant mixtures.

## **3.1. Gas chromatography**

196 Chromatography – The Most Versatile Method of Chemical Analysis

LAS, AES, AS 10-200 mL SPE C18+SAX

AEOs 50-2000 mL SPE C2+SCX+

**Method Solid** 

**phase** 

QAC 100 mL LLE **-** Solvent: chloroform (15 mL)

LAS, SPC 500-1000 mL LLE **-** Solvent: chloroform (3 x 4 mL)

NP1-2EO, NP, OP 300 mL LLE **-** Solvent: DCM (300 mL) [80] QAC 20 mL LLE **-** Solvent: chloroform (5 mL)

> (LAS) C2 (AES, AS)

> > SAX

QAC 50 mL SPE Alumina 1. Passing solution with SDS

NPEO, NPEC 100 mL SPE GBC 1. Conditioning: DCM, DCM/formic

LAS, SPC 25-250 mL SPE C18+SAX 1. Conditioning: MeOH, water

QAC 100 mL SPE Strata-X 1. Conditioning: ACN, water

**Table 3.** Overview of LLE and SPE techniques used for clean-up and preconcentration of surfactants

equilibrium extraction of the target compounds. Finally, the fiber is removed from the sample and extracted analytes are desorbed by diffusion into a different solvent (e.g., MeOH) [101]. This technique has been recently applied to isolation of cationic [101, 102], non-ionic [103] and anionic surfactants [104] from aqueous samples. Solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE) can be also considered for rapid isolation of surfactants. Both techniques are based on the diffusion of analytes from the sample directly, without requiring any organic solvent, into a fiber or bar made of a specific polymer. The amount of polymer changes from 0.5 µL in SPME fibers up to 300 µL

200 mL SPE C18 1. Conditioning: MeOH, water

**Isolation conditions Ref.** 

[82]

[84]

[79]

[85]

[92]

[95]

[89]

[52]

[96]

[55]

Washing: water Ion-par reagent: Patent Blue V

Ion-par reagent: methylene green

Ion-par reagent: disulphine blue

1. Conditioning: MeOH, water 2. Washing: MeOH/water 3. Elution: MeOH + HCl/MeOH 1. Conditioning: MeOH/isopropanol, water 2. Washing: water 3. Elution: MeOH/isopropanol

1. Conditioning: Not spec. 2. Fractionation: ACN 3. Fractionation: MeOH/ethyl acetate/water

2. Elution: MeOH

acid, MeOH, acidified water 2. Washing: MeOH/water, MeOH 3. Elution: DCM/formic acid

2. Washing: water, acidified water 3. Elution: MeOH + acidified MeOH

2. Washing: water 3. Elution: ACN/acetic acid/water

2. Fractionation: hexane/DCM 3. Fractionation: MeOH/DCM

**volume** 

**Analytes Sample** 

LAS, NPEO, NPEC, AEO, PEG, NP, OP

from environmental samples.

Less frequently used than HPLC for analysis of surfactants, the main drawback of GC is that all anionic and non-ionic compounds and their metabolites need to be derivatized with specific agents to solve sensitivity, separation or volatilization issues before injecting them into the system. Most commonly used derivatizing agents are trifluoroethanol [29, 58], diazomethane [84], N,O-bis(trimethylsilyl)trifluoro acetamide (BSTFA) [53, 59, 122], acetic anhydride [61, 109] and hydrogen bromide [90], among other reactants. In any case, some low molecular mass metabolites of non-ionic surfactants (NP and short-chain NPEOs) have been analyzed directly by GC [67, 80] as they are volatile enough, although better results can be

obtained if derivatization is performed. There are also some advantages in using GC over HPLC. Thus, GC columns have a better capability for achieving complete separation of homologues and isomers of many surfactants after derivatization. This may be a key aspect for those studies on the biodegradability or toxicity of surfactants such as LAS or NPEO, which can change depending on the length of the alkyl chain and/or the position of the phenyl ring [123] (Figures 3a and b). In most cases, anionic and non-ionic surfactants have been separated by nonpolar capillary columns containing 5%-phenyl-95%-dimethylpolysiloxane (e.g., HP-5 [58, 75, 67], SE-54 [76], DB-5 [84, 103, 109]), and a mobile phase comprised of high purity helium as carrier gas with a flow rate from 0.58 to 3.4 mL/min. Regarding cationic surfactants, the application of GC to their separation and analysis has not been mentioned in any paper so far [1]. Table 4 describes general information about some analytical protocols for determination of anionic and nonionic surfactants by means of GC in environmental samples.

Analysis of Surfactants in Environmental Samples by Chromatographic Techniques 199

**Column Detection LOD/** 

EI/CI(+)- MS

**MLD** 

0.8 ng/ mL

≤0.01 µg/L (LOQ)

0.01 mg/L

0.03 µg/L

0.01- 0.1 ng/L 0.08- 0.14 ng/g

EI(+)-MS 100 ng [67]

EI(+)-MS 0.002-

EI(+)- MS-MS

EI(+)-MS 0.16-

EI(-)-MS 0.001-

**Ref** 

[105]

[29, 143, 144]

[90]

[103]

[125]

anionic surfactants in water samples [124]. Nowadays, single quadrupole (MS) or tandem mass spectrometers (MS-MS) are commonly preferred because they allow unequivocal identification of analytes by measuring their parent masses and displaying specific fragmentation patterns after their ionization and rupture, respectively. Hence, there are several papers dealing with the analysis of anionic and non-ionic surfactants using GC coupled to MS [69, 105, 108] or MS-MS [125]. Target compounds can be detected by electron impact (EI) or chemical ionization (CI), being more widely used the first mode, although

higher sensitivity may be reached using CI for analysis of some anionic compounds.

**Recovery (%)** 

81-90 (NP) 75-112 (LAS, SPC)

**Mobile phase** 


65-102 Helium Rtx-1

(2 mL/min)

(1 mL/min)

(1 mL/min)

100 Helium

88.3-106.7 Helium

77-109 Helium

(capillary column, 30 m, 0.25 mm ID, 0.25 µm)

(capillary column, 30 m, 0.25 mm ID, 0.25 µm)

(capillary column, 60 m, 0.25 mm ID, 0.25 µm)

HP-5 (capillary column, 30 m, 0.25 mm ID, 0.25 µm)

DB-5 (fused silica capillary column, 30 m, 0.25 mm ID, 0.25 µm)

HP-5 (capillary column, 30 m, 0.25 mm ID, 0.25 µm)


**Target compounds** 

NP1-3EO, NP NP1-3EC LAS, SPC

> AEOs (C12-C15)

NP1-2EO, NP Marine

NP, OP River

NP,OP River

LAS Wastewa

ter, seawater

River water, wastewa ter

Wastewa ter, river water

sediment

water

water, sediment

**Matrix Sample** 

**preparation** 

Ion pair SPME Derivatization in GC injection port (tetrabutyl ammonium)

SPE (without derivatization) Derivatization (C3H7OH/ CH3COCl) Derivatization (SOCl2/ CF3CH2OH)

SPE Derivatization (HBr)

MAE, SPE (without derivatization)

DLLME Derivatization in situ (methyl chloroformate)

MAE, SPE Derivatization (BSTFA)

**Table 4.** Key aspects of GC analysis of surfactants in different environmental matrices.

Several types of detectors can be used after gas chromatography for the analysis of target compounds, such as flame-ionization detectors (FID), which were used for the analysis of

**Figure 3.** Selected GC-EI-MS characteristic ion chromatograms from a river sample, showing resolution of the derivatives of (a) LAS [145], and (b) NP and NP1EO (with their corresponding mass spectra) [144].

anionic surfactants in water samples [124]. Nowadays, single quadrupole (MS) or tandem mass spectrometers (MS-MS) are commonly preferred because they allow unequivocal identification of analytes by measuring their parent masses and displaying specific fragmentation patterns after their ionization and rupture, respectively. Hence, there are several papers dealing with the analysis of anionic and non-ionic surfactants using GC coupled to MS [69, 105, 108] or MS-MS [125]. Target compounds can be detected by electron impact (EI) or chemical ionization (CI), being more widely used the first mode, although higher sensitivity may be reached using CI for analysis of some anionic compounds.

198 Chromatography – The Most Versatile Method of Chemical Analysis

obtained if derivatization is performed. There are also some advantages in using GC over HPLC. Thus, GC columns have a better capability for achieving complete separation of homologues and isomers of many surfactants after derivatization. This may be a key aspect for those studies on the biodegradability or toxicity of surfactants such as LAS or NPEO, which can change depending on the length of the alkyl chain and/or the position of the phenyl ring [123] (Figures 3a and b). In most cases, anionic and non-ionic surfactants have been separated by nonpolar capillary columns containing 5%-phenyl-95%-dimethylpolysiloxane (e.g., HP-5 [58, 75, 67], SE-54 [76], DB-5 [84, 103, 109]), and a mobile phase comprised of high purity helium as carrier gas with a flow rate from 0.58 to 3.4 mL/min. Regarding cationic surfactants, the application of GC to their separation and analysis has not been mentioned in any paper so far [1]. Table 4 describes general information about some analytical protocols for determination of anionic and nonionic surfactants by means of GC in environmental samples. Several types of detectors can be used after gas chromatography for the analysis of target compounds, such as flame-ionization detectors (FID), which were used for the analysis of

**Figure 3.** Selected GC-EI-MS characteristic ion chromatograms from a river sample, showing resolution of the derivatives of (a) LAS [145], and (b) NP and NP1EO (with their corresponding mass spectra)

[144].


**Table 4.** Key aspects of GC analysis of surfactants in different environmental matrices.

## **3.2. Liquid chromatography**

High-performance liquid chromatography (HPLC) is currently the most commonly used technique for separation and analysis of commercial mixtures of surfactants in the environment, mainly due to its advantages over GC because HPLC is suitable for determining non volatile analytes from low to high molecular weight and derivatization is unnecessary in most cases. Reverse-phase columns, mainly RP-18 [47, 96, 106] and RP-8 [52, 95], are often employed for chromatographic separations of anionic, non-ionic, cationic surfactants and their degradation products. Mobile phases are solvent mixtures containing deionized water, acetonitrile and/or methanol. Separation can be improved by adding some additives (e.g., ammonium acetate (AMAC), triethylamine) to the mobile phase, as well as acetic (AA) or formic acid (FA) as modifiers [72, 71, 89]. There are also a few works showing efficient separation of NPEOs ethoxymers, some of their metabolites [126] and QACs [101] by amino-silica or cyanopropyl normal phase columns, although the elution order is reversed (more hydrophobic compounds, such as NP, elute first and NPEOs last). In these cases, stronger non-polar solvents (e.g., hexane, chloroform and isopropanol) are preferred. Additionally, some researchers have used new stationary phases that are specific for the separation of ethoxylated surfactants. As example, Lee Ferguson and co-workers [60, 127] tested a mixed-mode HPLC system using a column packed with a polymeric phase capable of separating NPEO and NP components by both size-exclusion and reversed-phase adsorption mechanisms (Figure 4a). Other authors have also applied this technique with some modifications to quantify OP and octylphenol ethoxylates (OPEOs) in environmental samples [73]. Alternative packing materials containing hydrophobic (alkyl chains) and hydrophilic (amide) functional groups to improve the simultaneous separation of cationic, anionic and non-ionic surfactants have also been occasionally employed [128].

Analysis of Surfactants in Environmental Samples by Chromatographic Techniques 201

interfaces that allow coupling HPLC to MS. Before this, mass spectrometry was used only for identification of a wide range of surfactants from their mass spectra by flow-injection

**Figure 4.** (a) Mixed-mode HPLC-ESI-MS total current ion chromatogram of NPEOs (A= NP, B=n-NP3EO, 0=NP, 1=NP1EO, etc.) from a sediment sample, switching MS polarity from positive to negative ion mode at retention time 25.8 min [60]; (b) UPLC-ESI-MS-MS extracted ion chromatograms showing the occurrence of NPEO metabolites in a sediment sample [44]; and (c) HPLC-MS-MS chromatogram of

a standard solution of QACs [141].

analysis (FIA) [134].

Some surfactant classes (e.g., LAS and NPEOs) and their metabolites are still good candidates, due to the presence of an aromatic ring in their molecular structure, to be analyzed by the first quantitative methods based on the use of HPLC coupled to ultraviolet (UV) or fluorescence detectors (FL) [68, 66, 126, 129, 130]. The presence of a benzene group also facilitates the use of UV for identifying some specific cationic surfactants such as benzalkonium chlorides (BACs) [97]. Moreover, HPLC coupled to FL detector was employed by Natkae and co-workers [131] to achieve partial separation of positional isomers and obtain information on the alkyl chain distributions of LAS in river water samples. However, aliphatic surfactants (e.g., AEOs and AES) have not been monitored so much due to their lack of UV absorbance or fluorescence. Prior derivatization using phenylisocyanate [132], naphthyl isocyanate and naphthyl chloride (NC) [88, 133], among others, must be carried out. Nowadays, however, this kind of surfactants, along with LAS, NPEOs and many other organic microcontaminants, are preferably determined by HPLC-MS, which offers several advantages over other detectors such as sensitivity, selectivity, and simultaneous identification and confirmation of multiple analyte classes by means of their molecular weight, retention time and mass spectra. In this sense, considerable progress has been achieved in the environmental analysis of surfactants over the last decade due to the development of atmospheric pressure ionization (APCI) or electrospray ionization (ESI) interfaces that allow coupling HPLC to MS. Before this, mass spectrometry was used only for identification of a wide range of surfactants from their mass spectra by flow-injection analysis (FIA) [134].

200 Chromatography – The Most Versatile Method of Chemical Analysis

High-performance liquid chromatography (HPLC) is currently the most commonly used technique for separation and analysis of commercial mixtures of surfactants in the environment, mainly due to its advantages over GC because HPLC is suitable for determining non volatile analytes from low to high molecular weight and derivatization is unnecessary in most cases. Reverse-phase columns, mainly RP-18 [47, 96, 106] and RP-8 [52, 95], are often employed for chromatographic separations of anionic, non-ionic, cationic surfactants and their degradation products. Mobile phases are solvent mixtures containing deionized water, acetonitrile and/or methanol. Separation can be improved by adding some additives (e.g., ammonium acetate (AMAC), triethylamine) to the mobile phase, as well as acetic (AA) or formic acid (FA) as modifiers [72, 71, 89]. There are also a few works showing efficient separation of NPEOs ethoxymers, some of their metabolites [126] and QACs [101] by amino-silica or cyanopropyl normal phase columns, although the elution order is reversed (more hydrophobic compounds, such as NP, elute first and NPEOs last). In these cases, stronger non-polar solvents (e.g., hexane, chloroform and isopropanol) are preferred. Additionally, some researchers have used new stationary phases that are specific for the separation of ethoxylated surfactants. As example, Lee Ferguson and co-workers [60, 127] tested a mixed-mode HPLC system using a column packed with a polymeric phase capable of separating NPEO and NP components by both size-exclusion and reversed-phase adsorption mechanisms (Figure 4a). Other authors have also applied this technique with some modifications to quantify OP and octylphenol ethoxylates (OPEOs) in environmental samples [73]. Alternative packing materials containing hydrophobic (alkyl chains) and hydrophilic (amide) functional groups to improve the simultaneous separation of cationic,

anionic and non-ionic surfactants have also been occasionally employed [128].

Some surfactant classes (e.g., LAS and NPEOs) and their metabolites are still good candidates, due to the presence of an aromatic ring in their molecular structure, to be analyzed by the first quantitative methods based on the use of HPLC coupled to ultraviolet (UV) or fluorescence detectors (FL) [68, 66, 126, 129, 130]. The presence of a benzene group also facilitates the use of UV for identifying some specific cationic surfactants such as benzalkonium chlorides (BACs) [97]. Moreover, HPLC coupled to FL detector was employed by Natkae and co-workers [131] to achieve partial separation of positional isomers and obtain information on the alkyl chain distributions of LAS in river water samples. However, aliphatic surfactants (e.g., AEOs and AES) have not been monitored so much due to their lack of UV absorbance or fluorescence. Prior derivatization using phenylisocyanate [132], naphthyl isocyanate and naphthyl chloride (NC) [88, 133], among others, must be carried out. Nowadays, however, this kind of surfactants, along with LAS, NPEOs and many other organic microcontaminants, are preferably determined by HPLC-MS, which offers several advantages over other detectors such as sensitivity, selectivity, and simultaneous identification and confirmation of multiple analyte classes by means of their molecular weight, retention time and mass spectra. In this sense, considerable progress has been achieved in the environmental analysis of surfactants over the last decade due to the development of atmospheric pressure ionization (APCI) or electrospray ionization (ESI)

**3.2. Liquid chromatography** 

**Figure 4.** (a) Mixed-mode HPLC-ESI-MS total current ion chromatogram of NPEOs (A= NP, B=n-NP3EO, 0=NP, 1=NP1EO, etc.) from a sediment sample, switching MS polarity from positive to negative ion mode at retention time 25.8 min [60]; (b) UPLC-ESI-MS-MS extracted ion chromatograms showing the occurrence of NPEO metabolites in a sediment sample [44]; and (c) HPLC-MS-MS chromatogram of a standard solution of QACs [141].

Among different types of mass analyzers used for the identification and quantification of surfactants, there are several authors that have employed single quadrupole HPLC-MS systems operating in selected ion monitoring (SIM) mode [25, 55]. However, isobaric interferences may lead to sensitivity and resolution issues, which have been commonly solved by means of triple quadrupole [27, 106, 135] or ion trap MS detectors [47, 94]. In recent years, both techniques, especially the first one, have been the main tool for trace analysis of surfactants and many other organic contaminants because their respective MS-MS (triple quadrupole) and MSn (ion trap) capabilities allow scanning for daughter ions, increasing sensitivity and selectivity (especially for analysis of environmental samples which contain compounds showing the same molecular ions and retention times than those for selected analytes) [72] (Figures 4b and c). As example, discrimination and quantification of the 20 positional isomers of LAS was achieved recently by Lunar and co-workers [136] by monitoring specific fragment ions resulting from the benzylic cleavage of the carbon alkyl chain on both sides of the LAS phenyl group. As a drawback of this type of MS detectors, there is a limited number of predetermined ions that can be monitored during a single experiment and, although less frequent than in single quadrupole MS, interferences may lead to overestimation in the concentration of target compounds. Time-of-flight (ToF) LC-MS systems are less commonly used than other MS analyzers for environmental analysis of surfactants, but their full scan spectral sensitivity in a wide mass range and accurate mass measurement allow the identification and quantification of a large number of target, nontarget surfactants and their metabolites in all kinds of matrices [40, 65, 137], constituting a recent alternative to address the issues mentioned above. Occasionally, hybrid systems like quadrupole time-of-flight (Q-ToF) detectors have been applied to determine a wide range of surfactants and some of their degradation products, such as alkylphenols and their carboxylates, in textile wastewaters [138], or to identify for the first time the molecular structure of LAS anaerobic degradation metabolites [139], although due to their high cost and relatively lower sensitivity compared with HPLC-MS-MS they are not often used for routine analysis of these compounds in environmental samples.

Analysis of Surfactants in Environmental Samples by Chromatographic Techniques 203

**Mobile phase Column Detection LOD/** 

Luna C18 (150 mm, 2mm, 5 µm)

LiChrosorb RP-8 (250 mm, 4.6 mm, 10.6 µm)

MSpak GF-310 4D filtration column (150 mm, 4.6 mm)

Luna C18 (150 mm, 2mm, 5 µm)

Purospher STAR RP-18 UHPLC column (50 mm, 2 mm, 1.8 µm)

Luna C18 (150 mm, 2mm, 5 µm)

Cyanopropyl column (250 mm, 2mm)

**MLD** 

0.1-2.6 ng/g (LOQ)

> 0.78- 37.3 ng/g

0.1-11.8 ng/L 0.1-23.7 µg/Kg

> <0.1- 27.3 ng/g

0.6-5 µg/Kg (LOQ)

µg/L

UV 0.7-5

FL 0.2-0.4 µg/L 5-10 µg/Kg

ESI(+)- ToF-MS

ESI-MS NPEO (ESI+) NP (ESI-)

ESI-ToF-MS LAS, SPC, NP1-2EC (ESI-) NPEO, AEO (ESI+)

> ESI(+)- MS-MS

> ESI(+)- MS-MS

**Ref** 

[65]

[52]

[127]

[40]

[44]

[27]

[101]

**Target analytes** 

BAC, DADMA C

> NPEO, NP

LAS, SPC, NPEO, NP1-2EC, AEO, PEG

AEO, NP1-3EO, NP, NP1-2EC

BAC, ATAC, DADMA C

**Matrix Sample** 

Marine sediment

marine sediment

Marine sediment, sewage

Sewage, marine sediment, seawater, s. solids

Marine sediment

River sediment, sludge

> water, sewage

QAC River

LAS, SPC Seawater,

**treatment** 

Sonication, LLE, SPE

Sonication, SPE LLE

Sonication, SPE

Sonication, SPE

> Soxhlet, LLE

Microporou s membrane liquidliquid extraction

**Recovery (%)** 

98-118 (DADMA C)

Soxhlet, SPE 75-105 MeOH/H2O,

64-127 (sediment)

26-117 (AEOs, PEGs) 60-108 (NPEOs, NP1-2EC) 37-101 (LAS, SPC)

ACN/H2O, isopropanol, FA, AMAC

H2O, tetraethyl ammonium hydrogensulfa te

H2O, MeOH, sodium acetate

ACN, H2O, FA, ammonium formate

FA, ammonium formate

isopropanol, FA, AMAC

34-88 ACN, H2O,

67-95 ACN/H2O,


**Table 5.** Key aspects of HPLC analysis of surfactants in different environmental matrices.

Table 5 provides general information about some analytical procedures aimed to the determination of surfactants in different environmental matrices by HPLC-MS (and some other detectors). So far, LAS and SPCs have been determined in both freshwater [71] and marine environments [40] using several kinds of MS detectors coupled to HPLC under negative ion (NI) mode due to the presence of a sulfonate group. Quasi-molecular ions [M-H] and a characteristic fragment m/z = 183 were used for their identification and quantification. AES have also been monitored in aquatic systems [62, 140] in a similar way, but m/z = 97, corresponding to HO – SO3- , was selected as the main fragment ion. On the other hand, identification of QACs relies upon measurement of their molecular ions (M+) in positive ionization mode (PI), and further confirmation can be achieved by mass measurement of main characteristic ions such as m/z = 60 for alkyltrimethylammonium chlorides (ATACs) [141] or m/z = 91 for BACs [94]. Non-ionic surfactants lack charge or acid/base functional groups, so the most widely used option for ionization of ethoxylated compounds, such as NPEOs and AEOs, is to form adducts as the oxygen atoms in the polyethoxylate chain can donate their free electrons to a selected cation agent and the flexible structure of the chain


routine analysis of these compounds in environmental samples.

corresponding to HO – SO3-

Table 5 provides general information about some analytical procedures aimed to the determination of surfactants in different environmental matrices by HPLC-MS (and some other detectors). So far, LAS and SPCs have been determined in both freshwater [71] and marine environments [40] using several kinds of MS detectors coupled to HPLC under negative ion (NI) mode due to the presence of a sulfonate group. Quasi-molecular ions [M-H] and a characteristic fragment m/z = 183 were used for their identification and quantification. AES have also been monitored in aquatic systems [62, 140] in a similar way, but m/z = 97,

identification of QACs relies upon measurement of their molecular ions (M+) in positive ionization mode (PI), and further confirmation can be achieved by mass measurement of main characteristic ions such as m/z = 60 for alkyltrimethylammonium chlorides (ATACs) [141] or m/z = 91 for BACs [94]. Non-ionic surfactants lack charge or acid/base functional groups, so the most widely used option for ionization of ethoxylated compounds, such as NPEOs and AEOs, is to form adducts as the oxygen atoms in the polyethoxylate chain can donate their free electrons to a selected cation agent and the flexible structure of the chain

, was selected as the main fragment ion. On the other hand,

Among different types of mass analyzers used for the identification and quantification of surfactants, there are several authors that have employed single quadrupole HPLC-MS systems operating in selected ion monitoring (SIM) mode [25, 55]. However, isobaric interferences may lead to sensitivity and resolution issues, which have been commonly solved by means of triple quadrupole [27, 106, 135] or ion trap MS detectors [47, 94]. In recent years, both techniques, especially the first one, have been the main tool for trace analysis of surfactants and many other organic contaminants because their respective MS-MS (triple quadrupole) and MSn (ion trap) capabilities allow scanning for daughter ions, increasing sensitivity and selectivity (especially for analysis of environmental samples which contain compounds showing the same molecular ions and retention times than those for selected analytes) [72] (Figures 4b and c). As example, discrimination and quantification of the 20 positional isomers of LAS was achieved recently by Lunar and co-workers [136] by monitoring specific fragment ions resulting from the benzylic cleavage of the carbon alkyl chain on both sides of the LAS phenyl group. As a drawback of this type of MS detectors, there is a limited number of predetermined ions that can be monitored during a single experiment and, although less frequent than in single quadrupole MS, interferences may lead to overestimation in the concentration of target compounds. Time-of-flight (ToF) LC-MS systems are less commonly used than other MS analyzers for environmental analysis of surfactants, but their full scan spectral sensitivity in a wide mass range and accurate mass measurement allow the identification and quantification of a large number of target, nontarget surfactants and their metabolites in all kinds of matrices [40, 65, 137], constituting a recent alternative to address the issues mentioned above. Occasionally, hybrid systems like quadrupole time-of-flight (Q-ToF) detectors have been applied to determine a wide range of surfactants and some of their degradation products, such as alkylphenols and their carboxylates, in textile wastewaters [138], or to identify for the first time the molecular structure of LAS anaerobic degradation metabolites [139], although due to their high cost and relatively lower sensitivity compared with HPLC-MS-MS they are not often used for

**Table 5.** Key aspects of HPLC analysis of surfactants in different environmental matrices.

allows the molecule to "wrap" itself around that cation [64]. Thus, sodium acetate [60, 74], ammonium acetate [89, 142] or different acids [53] are commonly added to the samples or to the mobile phase to increase the MS response of NPEOs and AEOs and to stabilize the generation of [M+Na]+, [M+NH4]+ or [M+H]+ ions, among others. Additionally, this ability to form different adducts can be used to obtain multiple confirmation points in full-scan mode [40]. Another advantage of MS compared to other detectors is that several types of surfactants can be analyzed within a single run (e.g., NPEOs and AEOs can be separated, using an adequate gradient, and later analyzed under PI [53, 64]). Most recent methodologies allow simultaneous determination of anionic and non-ionic surfactants and their metabolites in environmental samples [47, 55].

Analysis of Surfactants in Environmental Samples by Chromatographic Techniques 205

AA, Acetic acid; ASE, Accelerated solvent extraction; ACN, Acetonitrile; AEOs, Alcohol polyethoxylates; AES, Alkyl ethoxysulfates; AS, Alkyl sulfates; ATACs, Alkyltrimethylammonium chlorides; AP, Alkylphenol; APEOs, Alkylphenol polyethoxylates; APEC, Alkylphenol polyethoxycarboxylate; AMAC, Ammonium acetate; APCI, Atmospheric pressure ionization; BACs, Benzalkonium chlorides; BSTFA, N,Obis(trimethylsilyl)trifluoro acetamide; CWAX/TR, Carbowax/template resin; CI, Chemical ionization; DTDMAC, Dehydrogenated tallow dimethyl ammonium chloride; DADMAC, Dialkyldimethylammonium chlorides; DCM, Dichloromethane; DLLME, Dispersive liquidliquid microextraction; EI, Electron impact ionization; ESI, Electrospray ionization; EO, Ethylene oxide; CESIO, European Committee of Organic Surfactants and their Intermediates; FID, Flame-ionization detectors; FIA, Flow-injection analysis; FL, Fluorescence detectors; FA, Formic acid; GC, Gas chromatography; GBC, Graphitized black carbon; HPLC, High-performance liquid chromatography; HLB, Hydrophilic-lipophilic balance; LOD, Limit of detection; LOQ, Limit of quantification; LAS, Linear alkylbenzene sulfonates; LC, Liquid chromatography; LLE, Liquid-liquid extraction; MS, Mass spectrometry; MSPD, Matrix solid-phase dispersion; MeOH, Methanol; MLD, Method limit detection; MAE, Microwave-assisted extraction; NC, Naphthyl chloride; NI, Negative ionization; NP, Nonylphenol; NPEOs, Nonylphenol polyethoxylates; NPECs, Nonylphenol polyethoxycarboxylates; OP, Octylphenol; OPEOs, Octylphenol polyethoxylates; PA, Polyacrylate; PEGs, Polyethylenglycols; SDB, Polystyrene-divinylbezene; PDMS/DVB, Polydimethylsiloxane/divinylbenzene; PI, Positive ionization; PT, Potentiometric titrametration; PFE, Pressurized fluid extraction; PLE, Pressurized liquid extraction; Q-ToF, Quadrupole time-of-flight; QACs, Quaternary ammonium-based compounds; SIM, Selected ion monitoring; SDS, Sodium dodecyl sulphate; SLE, Solid-liquid extraction; SPE, Solid phase extraction; SPME, Solid phase microextraction; SBSE, Stir-bar sorptive extraction; SAX, Strong anionic-exchange; SCX, Strong cationic-exchange; SPCs, Sulfophenyl carboxylic acids; SFE, Supercritical fluid extraction; MS-MS, Tandem mass spectrometry; ToF, Time-offlight; UPLC, Ultra performance liquid chromatography; UV, Ultraviolet detectors; WWTPs,

**5. List of abbreviations** 

Wastewater treatment plants.

Juan M. Traverso-Soto, Eduardo González-Mazo and Pablo A. Lara-Martín

*Departamento de Química Física, Facultad de Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar (CEI∙MAR), Universidad de Cádiz, Puerto Real, Spain* 

[1] Thiele B (2005) Analysis of Surfactants in Samples of the Aquatic Environment. In: L.M.L. Nollet, editor. Chromatographic Analysis of the Environment. Boca Raton: CRC Press.

**Author details** 

**6. References** 

pp. 1173–1198.

Today, mass spectrometry is often combined with ultra-performance liquid chromatography (UPLC), which uses sub-2-µm column particles that provide enhanced separation, faster analysis, and improved sensitivity over HPLC, boosting laboratory efficiency by saving time and decreasing solvent consumption. Most researchers have started to benefit from this combination, although there are still a few examples on its use for analysis of surfactants. So far, UPLC-Q-ToF-MS has been used for structural elucidation of SPC isomers [139] and for environmental screening of several anionic and non-ionic surfactants in wastewater [138]. UPLC-MS-MS [44] has allowed achieving fast analysis (less than 10 min per sample) of NPEO metabolites and AEOs at trace levels in aquatic environments.
