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Modified method for the analysis of anionic surfactants as Methylene Blue

active substances

Article  in  The Analyst · July 1995

DOI: 10.1039/AN9952002001

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Rao Chitikela Steven Dentel

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Modified Method for the Analysis of Anionic
Surfactants as Methylene Blue Active
Substances
Srinivasarao Chitikela, Steven K. Dentel,* and Herbert E. Allen
Dcpartrncnt of Cil·il and L-m·ironmcntal f':nginecring, Unil'!:rsity of Delaware,
Newark, D£ 197fo, USA
Determination of anionic surfactants as Methylene Blue
active substances (MBASJ in water, waste water and sludge
samples is of increasing importance because of the rapidly
growing use of different types of anionic surfactants in
household and industrial applications. We have developed a
modified method for MBAS measurement, using sodium
dodecyl sulfate (SDS), an alkyl sulfate anionic surfactant
compound, as the reference anionic surfactant compound for
the MBAS analysis. The modified method has achieved a
reduction in sample size, elimination of the use of expensive
glassware, and a decrease in the quantity of chloroform used
compared with the standard method. The theoretical
relationship between the modified method and the standard
method was confirmed by the experimental calibration
curves. The linear calibration curves had r 2 values between
0. 994 and 0.999. On the basis of the statistical analysis on the
MBAS recoveries from standard addition and spiked
samples, the modified method is equally capable of extracting
anionic surfactant compounds from the samples, when
compared with the standard method.
Keywords: Methylene Blue acti1·c s11hstancc anionic
surfactant; water; specrrophotometr\'
Introduction
The analysis of anionic surfactants as Methylene Blue active
suhstanccs ( M BAS) is useful as a non-specific measurement of
the concentration of total anionic surfactants in water, waste
water. sludge and sediment samples. The method generally
used is that described in Standard Methods for the Examina-
tion of Water and Wastcwatcr. 1 Its usefulness is expected to
continue due to the increasing use of anionic surfactants in
household and industrial applications.:> The predominant
classes of anionic surfactants arc linear alkylhenzcne sulfo-
natcs and alkyl sulfates. hoth of which arc recovered
completely in the standard MBAS analysis. 1
Experience with the MBAS procedure' suggested the
desirability for a new method that would be less laborious,
require less laboratory space, eliminate the use of separatory
funnels and reduce the required quantities of chloroform (a
carcinogen that is also identified under the US Resource
Conservation and Recovery Act as a hazardous waste,-' and a
priority pollutant~). Several modified methods have been
developed for MBAS measurement in water, waste water and
sludge samplcs. 5 .<' Hevia ct a/5 5 modifcd the MBAS method
for sludge samples and influent and effluent waste water
samples. They used a 5, 10 or 15 cm-' sample volume and
conducted the MBAS analysis using half of all the reagent
volumes used in the standard method. Craig and Salvador<>
developed a micro-extraction method for MHAS analysis in
To\\ hom corre~pondcncc should he addrc:-,:-.cd.
Analyst, July 1995, Vol. 120 2001
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the range of 0-0.2 mg dm--'. They used reduced reagent
volumes and micro-extraction vessels ( 40 cm 3 screw-capped
dear borosilicate glass vials with PTFE-lincd silicone rubhcr
septa). Absorbancc at 652 nm was determined for the
extracted organic phasc-MBAS complex using a 5 cm optical
cell path length.
This paper reports a modified method for MBAS analysis
using glass culture tubes and Pasteur pipettes in place of
scparatory funnels, five-times less chloroform per analysis
than the standard method, and a reduction in lahoratory
space. The reference chemical compound used for this
analysis was sodium dodecyl sulfate (SDS), an alkyl sulfate
anionic surfactant. This was selected because of its extensive
use as a reference surfactant in research. The concentration
range for this MBAS analysis was 0.4-2.0 mg dm-> of SDS.
The MBAS analysis was conducted according to the standard
method and the modified method on the same standard
samples. The modified method's efficiency of recovering the
MBAS from the samples was determined by comparing with
the data obtained by the standard method.
Experimental
Reagent~·
SDS (about 99% purity, M, 288.4) was used as reference
MBAS compound and obtained from Sigma. St. Louis, MO,
USA. A 1 mg crn- 3 stock SDS solution was prepared ( IOO'Yo
active) and was stored in a refrigerator to avoid biodcgrada-
tion. A standard SDS solution was prepared by diluting 10 cm-'
of the stock SDS solution to I dm-' with de-ionized water.
Alcoholic phenolphthalein indicator solution was prepared
according to the standard method. Solutions of0.02 mol elm -'
NaOH and 0.01 mol dm- 3 H 2 SO~ were prepared. Chloroform
was obtained from Fisher Scientific, Malvern, PA. USA.
Methylene Blue reagent and wash solutions were prepared
according to the standard method. Glass wool was prc-
cxtracted with C'HCl 3 to remove impurities. The de-ionized
water used was determined to be MBAS-frce, and was used
for all reagent preparations and dilutions.
Apparatus
A Spectronic 21 (Milton Roy, Rochester, NY, USA) spectro-
photometer was used at a wavelength of 652 nm and with an
optical cell pathlcngth of 1 cm. Approximately 40 cm-'
round-bottomed borosilicate glass culture tubes with caps and
a Teflon fluorocarbon resin-faced rubber liner (Fisher Scien-
tific) were used for the modified procedure. and Pasteur
pipettes (23 cm long) were used for extraction of samples in
the analysis and were also ohtaincd from Fisher Scientific. A
vortex mixer (Fisher Scientific) was used for vigorous mixing
of working solutions in the modified MBAS analysis method.
2002 Analyst, July 1995, Vol. 120

Procedures 150 mm). The sample was adjusted to alkaline pH using

NaOH, with the pH exceeding 8.3 as indicated by the
A flow diagram of procedures used in the modified and
phenolphthalein in end-point. The pink colour from phe-
stand~rd ~nalytical methods, for comparison purposes, is
nolphthalein was then discharged by the addition of a few
given m Fig. 1. Standard solutions, as described below, were
drops of H 2 S0 4 . Then 2 cm3 of chloroform and 2 cm3 of
prepared and then used to generate three separate calibration
Methylene Blue reagents were added to each sample. The
cu~ves ~or the standard method procedure, and two separate
samples were shaken for 30 s using the vortex mixer. After
calibration curves for the modified method procedure.
allowing the phases to separate, the surfactant complex, along
with the chloroform in the organic phase, was transferred into
Modified method a second test-tube using a Pasteur pipette. This extraction
procedure was repeated twice on the solution in the first
Solutions of 0, 0.4, l.O, 1.5 and 2.0 mg dm--3 of SDS were
test-tube using 2 cm 3 of chloroform each time.
prepared using the standard SOS solution for each trial of
Then 10 em 3 of the wash solution was added to the second
calibration and analysis. Two solutions of standard additions
test-tube with the first phase extract (approximately 6 cm3).
of SOS and one spiked SOS solution were prepared for the
The tube was shaken for 30 s using the vortex mixer. After
measurement of percentage recoveries in the MBAS analysis,
settling of the contents, a Pasteur pipette was used to transfer
and are as follows: 0.27 mg of SOS in I dm3 of distilled water
the washed organic phase with MBAS into a third test-tube.
(to compare percentage recovery efficiency beyond the range
The solution in the second test-tube was extracted twice using
of 0.4--2.0 mg dm- 3 of MBAS) and 0.8 mg of SOS in 1 dm3 of
2 cm.1 chloroform each time. This washed extract of the
distilled water; and J .2 mg of SOS in 1 dm3 of tap water.
organic phase with MBAS (about JO cm3) was filtered through
A 5 cm 3 sample of each of the above solutions was used for
glass wool to transfer it into a fourth 20 cm3 marked test-tube.
the analysis (instead of JOO cm3, as used in the standard
The third test-tube was rinsed twice with 2 cmJ of chloroform
method). Each sample was placed in a test-tube (20 mm od x
each time. The chloroform was transferred into the 20 cm3
marked fourth test-tube, through the glass wool. The final
volume in the fourth test-tube was made up to 20 cm3 with
STANDARD METHOD MODIFIED METHOD
chloroform, making sure that all blue-coloured surfactant
100 ml sample in a 250 ml 5 ml sample in a 40 ml complex was transferred from the glass wool into the
separatory funnel: test-tube: add 0.01 mol dm-'
add 1 mol dm-3 NaOH until NaOH until alkaline test-tube. This 20 cm 3 final extract solution was used for
alkaline +
2 ml chloroform
measuring the absorbance at 652 nm using the spectropho-
+
10 ml chloroform + tometer. The same procedure was repeated for all standards
+ 2 ml Methylene Blue
25 ml Methylene Blue
and samples.

+ Procedure for the Standard Method

Wait for phase separation
The standard and modified method procedures are both
(i) Draw off chloroform layer with
MBAS into a second separatory
(i) Draw off chloroform layer with shown in Fig. J. The MBAS analysis of the standard and
MBAS into a second test-tube with a
funnel.
Pasteur pipette. spiked solutions was conducted according to the standard
(ii) Repeat the extraction of solution
(ii) Repeat the extraction of solution in the
in the first separatory funnel for two
first test-tube for two times with 2 ml
times with 1o ml chloroform each
chloroform each time.
time.

Add 10 mt wash solution to

the second test-tube Table_ 2 Percentage recoveries of standard and spiked SOS concentra-
twns m the standard method and in the modified method

Percentage recoveries(%)
(i) Draw off the washed chloroform
(i) Draw off the washed chloroform layer layer into the third test-tube with a SOS concentration Standard method Modified method
with MBAS into a 100 ml calibrated Pasteur pipette.
flask through a plug of glass wool. (ii) Repeat the extraction of solution in 0.27mgdm- 1 in
(ii) Repeat the extraction of solution in the second test-tube for two times with distilled water
the second separatory funne! tor two 2 ml chloroform each time.
times with 1O ml chloroform each
(iii) Transfer the total extract from the (standard addition) 90.9, 97.9, 95.8 95.4, 98.2, 90.0, 101.0
third test-tube into a fourth tesHube through 0.8mgdm Jin
time.
a plug of glass wool with thorough
(iii) Total solution in the calibrated flask rinsing of third test-tube. distilled water
to 100 ml marl< with chloroform. (iv) Total solution in the fourth test-tube to (standard addition) 92.7, 97.9, 96.7 94.8, 97.6, 100.3, 94.8
20 ml mark with chloroform.
1.2 mg dm-:i in tap
Measure absorbances of final extract
solutions at 652 nm against a blank
water (spiked
of chloroform. concentration on
0.03 mg dm-:i of
Fig. 1 Flow diagram of the procedures used in the modified and MBAS in tap water) 99.6, 98.3, 102.2 99.0, 98.0. 93.5, 102.4
standard analytical methods.

Table I Regression parameters for calibration curves of standard method and modified method

Standard method Modified method

Parameter Set I Set2 Sct3 Set I Set2

Intercept -0.003 -0.003 0.009 0.007 0.009
Slope 0.295 0.282 0.286 0.068 0.067
,2 0.996 0.994 0.999 0.998 0.998
St<mdard error of estimate 0.017 0.021 0.006 0.002 0.002
p value <(J.001 <0.001 <0.001 <0.001 <(l.001
Residual sum of squares 8.52 x ]()-'> 1.32 x I0 3 1.09 x j()-.J 4.37E x 10 " 3.59E x 10- 0

Table 3 Statistical analysis for percentage recoveries of standard and spiked SDS concentrations in standard and modified methods
Average ± I SE
Standard Modified
SOS concentration method method
0.27 mg dm-3 in distilled water 94.9±3.6 96.1±4.7
0.8 mg dm- 3 in distilled water 95.8 ± 2.8 96.8 ± 2.7
1.2 mg dm 3 in tap water IOO.O ± 2.0 98.3 ± 3.7
methods procedure I for the MBAS range of0.4-2.0 mg dm-3,
using 100 cm 3 samples. Although the standard method
recommends a volume increase to 250 cm 3 for a 0.27 mg dm-3
standard addition solution, the 100 cm3 volume was also used
in this case.
Results and Discussion
Calibration curves for the standard and modified methods
showed no significant curvature or other systematic deviation.
Linear regression analyses were performed to evaluate the
results. Table 1 shows the statistical values in the regression
analyses for all the calibration curves of standard and modified
methods. The standard errors of all the estimatcs7 are
relatively small. Specifically, they arc much less for the
modified method than for the standard method. The r2 values
ranged from 0.994 to 0.999 for both the methods. The residual
sum of the squares for all the calibration curves was either
(J.001, or less than 0.001. These values show that the linear
regressions arc very well fitted for the data. x The p values
(significance of F-statistic by the ANOV A) were also less than
0.001, which means that the linear fits are very highly
significant for the data. 9 A theoretical ratio (standard method
to modified method) of regression slopes of 4.04 was
calculated for these two methods based on the amount of
MBAS expected (due to SOS) in the chloroform solutions
(after accounting for chloroform solubility3). For example, the
MBAS concentration that has to be extracted in both the
methods at 2 mg of SDS per dm3 of sample is 2.02 µg of MBAS
per cm 3 of chloroform in the standard method and 0.5 µg of
MBAS per cm 3 of chloroform in the modified method
(including the necessary consideration of chloroform solubil-
ity in the different stages of analyses). Therefore, the
theoretical ratio of slopes of the calibration curves of both
methods is 4.04. The average regression slope for the standard
method's data was 0.2878, and similarly it was 0.0674 for the
modified method. Thus, an average experimental ratio of 4.27
for regression slopes of the calibration curves for both the
methods was observed, which deviated approximately 6%
from the expected 4.04.
Table 2 shows the percentage recoveries of standard and
spiked SOS concentrations in both the methods. The percen-
tage recoveries of standard and spiked concentrations of SOS
were satisfactory, most notably for the 0.27 mg SOS per dm3
of distilled water. This indicates that both the methods are
capable of measuring an MBAS value of 0.27 mg dm·-' in a
sample, which is beyond the accepted MBAS concentration
range of 0.4 to 2.0 mg of MBAS per dm-'. Table 3 shows the
average value ± standard crror, 9 and relative error10 of the
means for the percentage recoveries of MBAS in the analyses.
A 95% confidence interval based on the I-statistic included all
the points of all data sets. The result of a two-tailed F-test (for
comparison of the variance in the data sets of the two
methods) and a two-tailed /-test (for comparison of two
independent averages with unknown and equal variances) 11
on the two data sets of the two methods was that the variance
and the average values of the two methods were not
Analyst, July 1995, Vol. 120 2003
95% Confidence intervals Standard error ( % ) , relative
(I-statistic) error ('Yo). bias(%) of mean
Standard Modified Standard Modified
method method method method
94.9 ± 8.8 96.1±7.5 2.05. 3.75. 5.15 2.35. 4.9. 3.9
95.8 ± 6.9 96.8 ± 4.2 1.6. 2.9. 4.2 1.3. 2.7. 3.2
100.0 ± 4. 9 98.3 ± 5.8 1.15. 2.0. -0.03 1.8. 3.7. 1.75
significantly different at <Y = 0.05 significance level, which
means that the precision of the recoveries and the average
recovery values of the two methods were equivalent. The
statistical analyses demonstrate that the modified method has
comparable accuracy to the standard method for all the
standard and spiked MBAS analyses. Therefore, the modified
method developed in this work can be used for measurement
of anionic surfactants as MBAS in field samples, and is a
suitable alternative to the standard method.
Generally, the MBAS in sludge or sediment samples would
be analysed using the extract from methanol reflux with
anionic exchange processes, 1-' methanol-Soxhlct proccsses,5
or sublation processes, 1 from dried sludge or sediment
materials. The modified procedure developed in this research
would also appear applicable for the methanol extracts or
other extracts for MBAS measurements in various sludge or
sediment samples. Results using this revised method for
sludge and sediment samples will be reported elsewhere.
Conclusions
The modified method for MBAS analysis presented in this
work has several advantages over the standard method in
terms of the decreased sample size required, the decreased
volume of chloroform needed, and the reduction in laboratory
space required. It has an accuracy comparable to that of the
standard method. The calibration curves for the modified
method and the standard method showed the required
theoretical ratio between the slopes. This indicates that the
modified method is in fundamental agreement with the
procedure of the standard method. The regression coefficient
r2 values ranged from 0. 994 to 0. 999, demonstrating a high
correlation. The standard errors of estimates arc minimal in all
the analyses.
The modified method for MBAS analyses developed in this
work is an acceptable alternative to the standard methods. As
with the standard method, calibration and validation with a
laboratory standard is required. An interlaboratory compari-
son is recommended prior to its use in any formal protocol.
Funding support for this research from the USGS State Water
Research Institute Program is gratefully acknowledged.
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