ORIGINAL PAPER

Gas Chromatography with Flame Ionization Detectionof 1,4-Dioxane in Palm-Based Fatty Alcohol Ethoxylates

Bonnie Yen Ping Tay • Zulina Maurad •

Halimah Muhammad

Received: 13 August 2013 / Revised: 3 February 2014 / Accepted: 26 March 2014

� AOCS 2014

Abstract During the ethoxylation of fatty alcohol, 1,4-

dioxane, a toxic by-product may be formed. A simple and

rapid method using gas chromatography with a flame

ionization detector was developed for detection of 1,4-

dioxane in commercial palm-based fatty alcohol ethoxylate

(FAEO). The method involved spiking the 1,4-dioxane into

FAEO samples, and directly injecting the spiked samples

into GC with no clean-up steps. The method was validated

in terms of linearity, accuracy, intra-day precision and

inter-day precision, selectivity, limit of detection and limit

of quantification. In terms of linearity, a calibration curve

with a correlation coefficient of 0.9999 was obtained. The

accuracy of the method was indicated by recovery obtained

for spiked 1,4-dioxane samples at 5 levels of spiking, i.e. at

30, 60, 100, 200 and 500 lg/g, where recoveries were

within 99–105 % with relative standard deviation (RSD) of

\4.0 %. The RSD values of the intra-day and inter-day

precision were \1.0 %. The limit of detection and quan-

tification was 10 and 30 lg/g, respectively. The selectivity

of the method was indicated by its ability to analyze

commercial FAEO samples with different average moles of

ethylene oxide (EO). The GC profiles of these FAEO with

varying numbers of moles of EO were similar, and there

were no other peaks interfering with the 1,4-dioxane peak.

Keywords 1,4-Dioxane � Ethoxylation � By-products �Polyethoxylate � Method Development

Introduction

1,4-Dioxane with formula C4H8O2 (Fig. 1) is also known

as dioxane, 1,4-diethylene dioxide, p-dioxane or diethylene

ether. It is a heterocyclic organic compound appearing as a

clear, colorless liquid at room temperature and pressure. It

has a molecular weight of 88.11 g/mol, a density of

1.033 g/ml, a boiling point of 101.1 �C, and a melting

point of 11.8 �C. 1,4-Dioxane is classified as an ether, with

each of its two oxygen atoms forming an ether functional

group. It readily dissolves in water and is highly hygro-

scopic. It is a man-made compound that is not easily bio-

degradable. The toxicity of 1,4-dioxane was demonstrated

in studies which showed that laboratory animals developed

cancer after exposure to the lowest part per billion level

over the animal life time [1, 2]. Studies had also shown that

1,4-dioxane can be absorbed through excised human and

monkey skin [3, 4] and the intact skin of animals [5]. It is

known to have carcinogenic potential for humans as

reported by the US Department of Health and Human

Services, and International Agency for Research on Cancer

(IARC) [6].

During polymerization of ethylene oxide (EO) to pro-

duce polyethoxylate surfactants, such as polyethoxylated

alcohol and polysorbates, 1,4-dioxane can be formed as a

by-product of the combination and rearrangement of eth-

ylene oxide [7–9]. Another source of 1,4-dioxane is from a

further reaction of fatty alcohol ethoxylate with chloro-

sulfonic acid to form an alcohol ethoxy sulfate. Under this

acidic condition, the polyoxyethylene chain can be cleaved

and cyclized to 1,4-dioxane [7, 10]. Polyethoxylated raw

materials are widely used in cosmetic and personal care

(CPC) products as surfactants, emulsifiers, foaming agents,

and dispersants. 1,4-Dioxane may contaminate CPC pro-

ducts such as deodorants, shampoos, tooth pastes and

B. Y. P. Tay (&) � Z. Maurad � H. Muhammad

Advanced Oleochemical Technology Division, Malaysian Palm

Oil Board, No.6 Persiaran Institusi, Bandar Baru Bangi,

43000 Kajang, Selangor, Malaysia

e-mail: bonnie@mpob.gov.my

123

J Am Oil Chem Soc

DOI 10.1007/s11746-014-2471-9

mouthwashes if this ethoxylated product containing 1,4-

dioxane is used in the formulations.

Methods for analyzing low levels of 1,4-dioxane by gas

chromatography-flame ionization detector (GC–FID) has

been developed for polyethoxylated nonionic surfactants,

such as fatty alcohol ethoxy sulfate, PEG 150 distearate

[11] and commercial ethoxylated alkyl sulfates [12]. The

use of headspace solid phase microextraction coupled with

GC–MS for analysis of commercial polyethylene oxide,

poly(ethylene/propylene oxide) and polyhydric alcohol

nonionic surfactants, and cosmetics products were also

reported [13]. A method using GC/MS with selected ion

monitoring was developed for sodium laureth sulfate,

polysorbate 60 and PEG-8 [14]. A method that utilizes

HPLC with UV detection was developed for the assay of

1,4-dioxane in sulfated polyoxyethylene surfactants, i.e.

fatty alcohol ether sulfates and sodium laureth sulfate [15].

Most methods developed for assay of 1,4-dioxane focused

on sulfated FAEO. There is only one GC-FID method that

utilizes direct injection for detection of 1,4-dioxane in

petroleum-based FAEO or NEODOL� with EO contents

ranging from an average of 2.25 to 13 mol/mol of alcohol

[16].

This paper will report on the method developed for

detection of 1,4-dioxane in palm kernel oil-based FAEO by

GC-FID using a commercial capillary column with shorter

times of analyses.

Materials and Methods

Materials, Chemicals and Apparatus

Materials

FAEO samples with 3, 7 and 9 mol ethylene oxide (EO)

used as matrices for spiking were obtained from Thai

Ethoxylate Company Limited (Bangkok, Thailand). Other

FAEO (1, 2, 3, 5, 7, 9, 12 and 20 mol EO) were obtained

from local and overseas commercial companies.

Standard

1,4-Dioxane (99.0 % min purity) used as the calibration

standards was a reagent-grade chemical from Fisher Sci-

entific (Waltham, MA, USA).

Chemicals

HPLC-grade acetonitrile (99.9 % purity) was obtained

from QREC Chemical Co Ltd. (Auckland, New Zealand).

Apparatus

Duran� volumetric flasks (10 ml) were purchased from

Schott Ltd (Mainz, Germany). Volumetric flasks (5 ml)

Pyrex A ISO 1042, BS1792 were obtained from SciLab-

ware Ltd (Staffordshire, UK). The electronic dispenser,

Multipette stream � and 2.5 ml combitips were bought

from Eppendorf (Hamburg, Germany). A vortex mixer was

obtained from Vision Scientific Co. Ltd (Gyeonggi-do,

South Korea) while microvials were obtained from Agilent

Technologies (Palo Alto, CA, USA).

Method

Standard Stock Solutions and Calibration Curve

The initial stock solution of 1,4-dioxane standard

(&1,000 lg/ml) was prepared by dissolving about 0.01 g

of the analyte in HPLC grade acetonitrile in a 10-ml vol-

umetric flask. From the stock solution and subsequent

dilutions, a range of 1,4-dioxane concentrations were pre-

pared at 0.5, 1, 5, 10, 30, 50 and 70 lg/ml, and were

analyzed by a GC–flame ionization detector (FID) to pre-

pare the calibration curve.

Gas-Chromatography-Flame Ionization Detector

(GC–FID)

The Agilent 7890A GC–FID with a DB-5 fused silica

capillary column coated with a 1-lm polysiloxanes sta-

tionary phase (30 m length 9 0.32 mm i.d.) was used for

analysis of 1,4-dioxane. The carrier gas was helium and set

at constant flow of 0.8 ml/min. The hydrogen flow was

maintained at 30 ml/min, air flow at 300 ml/min and make-

up flow (nitrogen) was 25 ml/min. A split ratio of 10:1 was

used. The detector temperature was set at 310 �C. The inlet

temperature and pressure were 200 �C and 4.47 psi,

respectively. The oven temperature was set at 50 �C, and

held for 4 min, then increased at a 10 �C/min rate to

Fig. 1 Structure of 1,4-dioxane

J Am Oil Chem Soc

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110 �C and held for another 20 min. The total time for one

run was 24 min. A post run after one cycle analysis was

performed at 300 �C for 10 min. A focus liner 4 mm (i.d.)

with glass wool (part no. 092002) SGE Analytical Science

(Victoria, Australia) was used.

GC–MS Analyses

The GC–MS used was from 7890A Series Hewlett-Packard

(Agilent Technologies, CA, USA) fitted with 5975C Triple-

Axis mass spectrometer. Data handling and system opera-

tions were controlled by the GC–MS NIST05 software.

Separation was carried out using an Agilent 19091 J-413

HP-5 column (30 m length 9 0.25 mm i.d., 0.25 lm film

thickness) with helium (99.99 % purity) as the carrier gas at

flow rate of 0.74 ml/min. The injector temperature was kept

at 200 �C in a split mode of 10:1 with a split flow of 20 ml/

min at 1 min, and a total flow of 23.7 ml/min. The inlet

pressure was 4.47 psi. The oven temperature programming

was: initial temperature at 50 �C (held for 4 min), ramped at

10 �C/min to 110 �C (held for another 20 min). The full

scan of electron ionization mass spectra was obtained under

the following conditions: mass-to-charge ratio (m/z) scan

range from 30.0 to 500.0, and solvent delay was 3.0 min.

The GC–MS interface and ion source temperature were set

at 150 and 230 �C, respectively.

Preparation of Untreated and Spiked Fatty Alcohol

Ethoxylate for Recovery Study

The stock solution of 1,4-dioxane in acetonitrile

(10,000 lg/ml) was diluted to the appropriate working

solutions for spiking. (Four replicates were analyzed for

each of the five spiking levels 30, 60, 100, 200 and 500 lg/

g). Spiked and blank samples were injected directly into

the GC-FID.

Results and Discussion

Method Development and Structure Confirmation

The first focus of this work was to optimize the GC-FID

conditions so that it will allow separation of 1,4-dioxane

from peaks arising from the heavier ethoxylate matrix,

FAEO. The previously reported direct injection method for

analysis of Neodol FAEO was carried out using a dual

columns system with column switching to allow separation

of 1,4-dioxane from interfering peaks from the ethoxylates

and other polar volatile components [14]. Their earlier

attempts at direct injection using single GC columns were

not successful in separating 1,4-dioxane from interfering

components [14]. In this work, a DB-5 GC column was

used. The GC condition was optimized for detection of 1,4-

dioxane in HPLC-grade acetonitrile. The solvent peak and

its impurities did not interfere with the 1,4-dioxane peak.

Then the ethoxylate sample free from 1,4-dioxane was

injected directly, and the resultant chromatogram showed

no overlapping peaks with the retention time of 1,4-diox-

ane. Therefore, unlike the HPLC method for sulfated

FAEO which used solid phase extraction to clean up matrix

peaks, these samples can be analyzed directly without

sample preparation [15]. A split focus liner with deacti-

vated glass wool was used at the inlet port, and replaced if

contaminated over time. The glass wool in the liner is able

to trap the heavier ethoxylates and only allow the volatiles

through. This method is a direct injection method and over

time other residues may accumulate in the column after

repeated analyses. Therefore, an additional post run for

10 min at 300 �C was included after every analysis to

remove other volatile residues from the column. The

absence of 1,4-dioxane in FAEO used as the matrix for

spiking was confirmed through comparison of chromato-

grams of unspiked FAEO, and 1 lg/ml of 1,4-dioxane in

acetonitrile (Fig. 2). The instrument limit of detection for

1,4-dioxane in acetonitrile was 1 lg/ml where its signal to

noise ratio (S/N) [ 3 [17]. It was found that no peaks

interfered with 1,4-dioxane for the unspiked FAEO, and

therefore these FAEO can be used as the matrix for spiking.

Confirmation of the presence of 1,4-dioxane in FAEO was

obtained by GC–MS analysis using similar conditions as

the GC-FID method using an Agilent Technologies HP5-

MS column. The mass spectra of spiked 1,4-dioxane

(50 lg/ml) in FAEO is shown in Fig. 3. 1,4-Dioxane was

detected at 4.753 min. The match of 1,4-dioxane mass

spectra from the experiment and the library was 94 %.

Therefore, the identity of the 1,4-dioxane was confirmed in

FAEO.

Method Validation Parameters

The method was validated in accordance with the param-

eters as described in the International Conference on Har-

monization (ICH) Guidelines [18]. The method was

validated in terms of linearity, accuracy, precision, selec-

tivity, limit of detection and limit of quantification.

Recovery/Accuracy

The accuracy of the method was determined by spiking

1,4-dioxane into FAEO. FAEO with three different num-

bers of moles of EO used as the spiking matrices. The

results in Table 1 show recoveries of 96–105 % for all

spiking levels for all three matrices, which met our labo-

ratory’s criteria of 80–120 % recovery. Replicate analyses

yielded RSD of \5 % for all levels.

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Fig. 2 GC chromatograms of a 1,4-dioxane standard solution (1.0 lg/ml), b blank FAEO sample and c spiked FAEO sample (1.0 lg/ml)

J Am Oil Chem Soc

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Calibration Curve and Linearity

The linearity of the detector response was checked within

the working range of 0.5–70 lg/ml of 1,4-dioxane standard

used for external calibration. For each of the concentration

levels, six individual replicates were injected. The cali-

bration curve can be expressed by the least square regres-

sion equation y = 0.6524x [correlation coefficient

(R2) [ 0.9999 and standard error of 0.05] where y is the

GC detector response measured as peak area and x is

concentration (lg/ml) of 1,4-dioxane standard. The R2

showed that the calibration curves have good linearity

within the working range.

Limit of Detection and Quantification

The limit of detection (LOD) is the lowest concentration

of analyte in a sample that can be detected, but not nec-

essarily quantified under specified experimental conditions

[19] and was determined at a signal-to noise (S/N) ratio of

3:1. In this study, four replicates of spiked FAEO at the

lowest detectable concentration (0.5 and 1.0 lg/ml) were

analyzed, and it was found that LOD was 1 lg/ml (S/

N [ 3). Figure 3 shows the comparison of chromatograms

of 1,4 dioxane standard (1 lg/ml), blank and FAEO

(spiked with 1 lg/ml 1,4-dioxane). 1,4-Dioxane in aceto-

nitrile was detected at 8.59 min. The limit of quantifica-

tion (LOQ) was based on a S/N of 10:1, which is the

lowest concentration of the analyte which can be

Fig. 3 Mass Spectra of spiked

1,4-dioxane in FAEO

Table 1 Percentage recovery

of 1,4-dioxane from palm-based

fatty alcohol ethoxylates with

different numbers of moles of

ethylene oxide

Concentration of 1,4-

dioxane (lg/g)

FAEO (3 mol EO) FAEO (7 mol EO) FAEO (9 mol EO)

Recovery (%)

n = 6

RSD

(%)

Recovery (%)

n = 6

RSD

(%)

Recovery (%)

n = 6

RSD

(%)

30 103.5 1.5 99.9 2.4 96.8 2.3

60 101.4 2.5 100.0 2.4 98.4 2.9

100 99.9 1.3 98.0 1.5 96.9 3.3

200 101.8 2.2 97.3 2.9 96.2 1.9

500 104.3 3.0 96.5 3.8 97.2 2.1

Table 2 Intra-day precision results and statistical data

Concentration of 1,4-dioxane (lg/

ml) n = 4

Percentage recovery

(%)

RSD

(%)

3.1 99.4 0.4

15.5 100.6 0.4

50.5 101.4 0.2

Table 3 Inter-day and intermediate precision data and statistical data

Inter-day and intermediate precision for 1,4-dioxane at

10 lg/ml n = 6

RSD

(%)

Day 1 0.5

Day 2 0.4

Analyst 1 0.5

Analyst 2 0.4

J Am Oil Chem Soc

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measured repeatedly and accurately by the method. In this

study, 3 mg/ml 1,4-dioxane has an S/N of above 10 (six

replicates determination of spiked FAEO). Therefore, the

LOQ of the method is 3 lg/ml and the accuracy data from

recovery was good at this spiking level as shown in

Table 1.

Fig. 4 GC-FID chromatogram of: a 3 lg/ml 1,4-dioxane, b FAEO, 1 mol EO, c FAEO, 5 mol EO d FAEO, 12 mol EO, e FAEO, 20 mol EO.

EO peak not detected in chromatogram

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Repeatability

The intra-day precision (repeatability) of the developed

method was determined by analyzing a 10 lg/ml standard

solution (six replicates), and also at 3 lg/ml (low), 15 lg/

ml (medium) and 50 lg/ml (high) concentrations (four

replicates each) using the same equipment, and performed

by the same analyst. For analyses of the 10 lg/ml standard

solution, the relative standard deviation (RSD) of the

average calculated concentration was between 0.5 and

1.0 % for the three levels of concentration (Table 2). Inter-

day precision was determined by analyzing six replicates of

10 lg/ml standard over 2 days. The RSD for inter-day

precision was 0.5 and 0.4 % for day 1 and day 2 of analyses,

respectively. Intermediate precision was performed by

analyzing 6 replicates of 10 lg/ml standard solution pre-

pared by two different analysts. The RSD for intermediate

precision RSD obtained did not exceed 0.5 % (Table 3).

Determination of 1,4-dioxane from commercial palm-

based FAEO

Selectivity of the method is part of the validation procedure

to confirm the ability of the method to accurately assess the

analyte in the presence of other components such as

impurities, degradation products or from the matrix [19].

The selectivity of the method was evaluated by analyzing

1,4-dioxane in commercial palm-based FAEO with average

numbers of moles of EO ranging from 1 to 20. A total of 19

samples were analyzed in triplicates and none of them were

found to contain 1,4-dioxane at the detectable value of the

GC-FID. Figure 4 shows some representative chromato-

grams of commercial FAEO with average numbers of

moles EO of 1, 2, 5, 12 and 20 and the 1,4-dioxane stan-

dard in acetonitrile. Ethylene oxide was not detected using

this GC-FID condition. The chromatograms were found to

show similar profile, and therefore method developed can

be applied for these FAEO with different numbers of moles

of EO.

Conclusion

A GC-FID procedure with direct injection method was

developed to determine the presence of 1,4-dioxane in

palm-based FAEO. This method was also validated in

terms of LOD, LOQ, accuracy, repeatability/reproduci-

bility and selectivity. The presence of 1,4-dioxane in the

spiked FAEO was confirmed using GC–MS. This method

can be applied for the analyses of 1,4-dioxane in palm-

based FAEO with varying numbers of moles of EO as

matrix interference was minimal due to the use of a liner

with glass wool at the inlet injection of GC.

Acknowledgments The authors wish to thank the Director General

of MPOB for permission to publish this paper and Thai Ethoxylate

Company Limited, Bangkok, Thailand for providing fatty alcohol

ethoxylate samples used as matrices during the recovery work.

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