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Micro-determination of arsenic in aqueous samples by image scanning and

computational quantification

Article in Analytical methods · January 2012

DOI: 10.1039/c1ay05569k

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 Analytical Dynamic Article Links < C
Methods
Cite this: Anal. Methods, 2013, 4, 242
www.rsc.org/methods PAPER
Micro-determination of arsenic in aqueous samples by image scanning and
computational quantification
Muhammad Salman,* Makshoof Athar, Waheed-uz-Zaman, Umer Shafique, Jamil Anwar, Rabia Rehman,
Sadia Ameer and Muhammad Azeem
Received 10th September 2013, Accepted 10th November 2013
DOI: 10.1039/c1ay05569k

Arsenic is highly toxic in all of its forms found in natural groundwater. An improved method for the
estimation of inorganic arsenic at low levels (mg L—1) in water has been proposed. The method involves
the generation of arsine in a specially designed cell by borohydride reduction of arsenite (AsO2 1—). The
resulting arsine is passed through a filter paper pre-dipped in mercuric bromide solution giving
a yellowish brown complex. The color intensity of the spots is calculated by scanning the spotted paper
and analyzing the image using specially designed software. The method was found to be effective at
trace levels having a linear response at the concentration range 2–20mg L—1 (8–80ng). The detection
limit of the proposed method is 1 mg L—1 (4 ng) which can be reduced further by making some
modifications to the apparatus. The method was successfully applied to the analysis of synthetic
samples and field samples of water.

Introduction and easy to perform.17,18 The major drawback is that the

Arsenic occurs in nature in both inorganic and organic forms and
the contamination of drinking water by arsenic is a worldwide
problem. The main sources of arsenic in water are geochemical
materials which release arsenic under geochemical conditions
naturally.1 The permissible arsenic level in drinking water and as
designated by the U.S. Environmental Protection Agency and
the World Health Organization is 10 ppb (0.01 ppm). 2 Thus, the
arsenic content in industrial raw materials, product effluents as
well as in drinking water supplies should be monitored with
precision and reproducibility.3
Several analytical techniques have been employed for arsenic
determination at low levels. Most of the employed techniques
involve sophisticated/expensive techniques like Atomic Absorp-
tion Spectrometry (AAS),4,5 Inductively Coupled Plasma-Optical
Emission Spectroscopy (ICP-OES),6 UV/VIS Spectrometry,7–10
HPLC11 etc. Some other techniques which are not common in
routine analysis have also been employed for arsenic determi-
nation at very low levels. These include amperometry, 12 neutron
activation analysis,13 voltammetry,14,15 X-ray fluorescence16 etc.
Another analytical technique which has been employed for
arsenic determination at microlevels is the spot test, a well known
Gutzeit Method. It includes the complex formation of arsine
generated from arsenic containing solution with mercuric
bromide. The advantage of this technique is the elimination of
expensive instruments and that it can be employed in kit form for
the analysis of arsenic. Moreover, the method is sensitive, rapid
Institute of Chemistry, University of the Punjab, Lahore, 54590, Pakistan.
E-mail: salmans_rajpoot@yahoo.com
measurement of the colored complex involves the naked eye
which leads to poor repeatability and a considerable determi-
nation error of 20–35%. Efforts are being made to make this
method reproducible and precise. 19
The aim of this work is a contribution towards these efforts. In
this work a modified apparatus has been suggested for arsine
generation and spot fixation. Moreover, the densities of the spots
were measured using a flatbed scanner connected to a computer
having specially developed software. The method presented is
simple, portable, economical, accurate, and easy to use in field
research.
Experimental
Reagents and apparatus
Sodium arsenite (East Anglia Chemicals), mercuric bromide
(Fluka), sodium borohydride (Acros) and HCl (Merck) were
used in the study. Watman filter paper No. 42 was used to
prepare the reagent paper and a UV/Vis Spectrometer (Labomed
UVD-3500) was employed to quantify arsenic by the conven-
tional method. A reflective flatbed scanner (BENQ 5000 L) was
used for image scanning in the proposed method.
Stock solution of arsenic
A stock solution of arsenic (1000 mg L—1) was prepared by
dissolving an appropriate quantity of extra pure sodium arsenite
(NaAsO2). Standard solutions of required concentrations
(2–20mg L—1) were prepared by successive dilution of this stock
solution.

242 | Anal. Methods, 2013, 4, 242–246 This journal is ª The Royal Society of Chemistry 2013
Preparation of reagent paper
Watman filter paper No. 42 was dried to remove moisture and
then soaked in the solution of mercuric bromide in a petri dish
for 15 mins. After soaking it was then dried and used for arsenic
quantification. Lead acetate paper was also prepared in the same
fashion.
Color density measurement
A reflective flatbed color scanner (BENQ 5000 L) was used to
acquire the spot images. A ‘Visual Basic’ graphical application,
specially developed for this purpose, was then used to calculate
the cumulative color density of a predefined circular area of the
spot. A fixed area was analyzed each time during the run.
Assembly for arsine (AsH3) generation and spot fixing
A schematic hydride generation assembly was fabricated that
can convert soluble arsenic to volatile arsine (AsH 3) and can
guide the liberating arsine to pass through a reagent paper. The
hydride generation assembly is shown in Fig. 1. The assembly
consists of a pyrex glass tube having a capacity of 8 mL. The
reaction tube has an acid inlet covered with a self sealing rubber
stopper and an outlet for the guidance of arsine. The reaction
tube is also fitted with a gas bubbler through which nitrogen gas
can be bubbled in the reaction mixture in order to complete the
removal of arsine from the mixture and to avoid stirring. The
outlet of the reaction tube is passed through a teflon holder
containing a reagent paper designed especially for this purpose.
The holder was designed in such a way that the liberated arsine
passes only through the center of the paper and the spots
produced are of equal size every time. To avoid any leakage of
the gas, rubber O-rings were screwed tightly to the outlet of the
reaction tube and also within the holder. A needle valve flow
controller was used to maintain a uniform flow of nitrogen and
a glass manometer was used to measure the pressure of the
nitrogen.
The same assembly can be used for samples having extremely
low concentrations of arsenic in large volumes, just by
replacing reaction tube with a round bottomed septum flask
(100–250 mL).
Fig. 1 Assembly of arsine generation and spot fixation.
This journal is ª The Royal Society of Chemistry 2013
Methodology
To start, 4 mL of arsenic standard solution (2 mg L—1; 8 ng As)
was added to the reaction tube followed by the addition of
hydrochloric acid (5 M, 2 mL). The assembly was then sealed as
shown in Fig. 3.1. Nitrogen gas was then turned on to ensure the
proper mixing of the solution components. 2 mL of sodium
borohydride (5%) was injected drop wise through an injection
branula no. 18 equipped with an injection syringe. As soon as
sodium borohydride entered the reaction mixture arsine gas was
liberated, this passed through the outlet which was equipped with
a teflon holder containing reagent paper. The reagent paper was
prepared by impregnating the circular disc (2 cm diameter) of
Whatman filter paper No. 42 with freshly prepared mercuric
bromide solution (2%) and then drying. A simple filter paper
formerly dipped in lead acetate solution was placed between the
passage of arsine and the reagent paper in order to eliminate any
sulfide interference. A brownish yellow colored spot appeared on
the paper. The reaction was continued for 3–5 min for complete
liberation of arsine. The color density of the spot appearing on
the reagent paper was then calculated by importing the spot
image to the computer using a flat bed scanner (BENQ 5000L)
and analyzing with densitometry software; a ‘‘Visual Basic’’
graphical application, specially developed for this purpose.
Different process parameters such as the effect of concentra-
tion of arsenic solution, concentration of acid, concentration of
mercuric bromide and time of reaction were studied to find the
optimum working conditions. Fresh arsenic standards (8–80 ng)
were analyzed, and a calibration line was plotted. Standard error
of estimates, correlation coefficient, and other statistical
parameters were calculated. In addition, synthetic arsenic
samples were tested to evaluate the accuracy of the technique.
The method was also successfully applied to field samples of
water.
Results and discussion
Quantification of arsenic in environmental samples is a sensitive
issue. Several methods have been employed for quantification of
arsenic in environmental samples. But these methods are either
costly or not precise at low levels. Efforts are being made around
the world to develop accurate, inexpensive, portable and rela-
tively easy methods to quantify this toxic pollutant. The present
work is a contribution towards these efforts.
The term ‘‘spot test analysis’’ means sensitive and selective
detection methods that are based on chemical reactions in which
an essential feature is to employ a drop of the analyte on the
reagent(s). The detections are micro-analytical and applicable to
a wide range of organic and inorganic compounds. In general,
the spot test procedure is simple and rapid. Efforts have been
made to quantify the process. The present study is also a contri-
bution in this field as the technique is simple and cost-effective
can be used as on-field analysis.
In previous designs of apparatus for measuring arsenic by spot
test, either a horizontal reaction tube was used or a tube inclined
at an angle.19 Such a design has a serious drawback in that the
reagent paper may be destroyed by the reaction mixture which
has been previously removed by using a long capillary tube as
used in the conventional Gutzeit method. But it introduces
Anal. Methods, 2013, 4, 242–246 | 243
another drawback i.e. low sensitivity due to the increase in dead
volume (the empty area where the generated arsine may be
retained and effect precise quantification). In this study, the
apparatus has been modified in such a way as to remove these
drawbacks and increase sensitivity and reproducibility (Fig. 1). A
nitrogen purging system is involved to reduce the dead volume
effect and to ensure the proper mixing of the reaction contents
thereby ensuring precise quantification. Another previous design
containing a two section reaction tube filled with resin has been
proposed for field analysis 20 but the effect of dead volume was
not taken into consideration.
In the proposed method, inorganic arsenic ions have been
estimated indirectly, that is, by interacting ions with nascent
hydrogen (produced in situ using sodium borohydride and
hydrochloric acid) and converting them to arsine (AsH 3). The
resulting arsine gas is passed through the filter paper having lead
acetate in its pores, where any sulfide impurity (in the form of
H2S after reacting with HCl) gets reacted with lead acetate to
produce lead sulfide (PbS). Finally the gas is allowed to pass
through the reagent paper (Watman filter paper No. 42 pre-
impregnated with mercuric bromide), where it reacts with
mercuric bromide to form a yellow colored arsenic-mercuric
bromide complex. The more arsenic ions are present in the
solution the greater the amount of arsine produced and the
greater the amount of As(HgBr) 3 complex is produced on
the reagent paper. As a result, the spots on the paper will become
darker and darker with an increase in arsenic ions. Therefore, the
As(HgBr)3 spots are quantifiable. A flow of nitrogen was used to
decrease the effect of dead volume in the reaction flask/tube.
However, the pressure of nitrogen should not be high, as it will
rupture the paper. Sulfuric and hydrochloric acids were used to
produce an acidic medium in the reaction, but the former created
alot of heat when added to aqueous solutions of arsenic and the
lead acetate-dipped paper burnt because of the rise in tempera-
ture of the passing gas. As a result, HCl was selected for all the
experiments.
The quantification of arsenic spots was carried out using
specially developed software. A digital image of the medium
containing the developed spots was saved to a PC using a flatbed
image scanner at 300 dpi, 24-bit RGB, reflective mode. The
software measured the red, green, and blue parts of each pixel of
the spot and summed up the values to give a final color depth.
The RGB color space is a subtractive color presentation: a pure
white color would mean that each of components is at their
highest value (eight bits per component ¼ 255) and a pure black
color would have each red, green, and blue component equal to
zero.21,22 For instance, in Fig. 2, the color density of the sixth spot
(12 ppb arsenic concentration) was 107.05, which had 7.27 units
red (6.79%), 32.38 green (30.24%), and 67.40 blue (62.97%).
The effects of different parameters such as acid concentration,
reaction time, sodium borohydride concentration, which may
Fig. 2 Spots with variable arsenic concentration 2–20 ppb (8–80 ng).
244 | Anal. Methods, 2013, 4, 242–246
affect the color intensity of the spot, were also checked to find the
optimal conditions. A series of solutions with varying acid
concentrations (1–5 M) was prepared for studying the effect of
acidity. Fig. 3. demonstrates the response as a function of acid
concentration. The higher the HCl concentration, the more dense
the spot on the reagent paper. So, it can be concluded that
concentrated HCl can be used more effectively but should be
used carefully because the reaction becomes so exothermic and
extensive bubbling in the reaction tube was observed. Fig. 4
illustrates the color density as a function of reaction time. The
spot intensity was found to increase continuously up to 4 min of
reaction time after that there was no effect on spot intensity
indicating that a 5 min reaction time would be enough for
completion of the reaction. The effect of sodium borohydide
concentration was also studied by varying its concentration (1–
5%). It was found that there is no significant change in the color
density of the spot due to variation in sodium borohydride
concentration. A similar effect was noted for the variation in
mercuric bromide concentration (0.5–2.5%).
A calibration line for the estimation of arsenic was drawn with
the concentration of arsenic as abscissa and the color density of
the spots as ordinate (Fig. 5). The experiments for obtaining
calibration were performed at optimized conditions. A correla-
tion analysis was carried out to obtain the coefficient of deter-
mination or correlation of coefficient (R2). The value, 0.992
approaching to one points out the goodness of fit for the
experimental data (Table 1). The detection limit for the experi-
ment comes out to be 1 ppb aqueous arsenic concentration. The
calibration data indicates a wide linear range i.e. 2–20 ppb (8–80
ng) arsenic concentration. It may extend up to 50 ppb (if needed).
Beyond a concentration of 50 ppb arsenic the color of the spot
Fig. 3 Color density as a function of HCl concentration.
This journal is ª The Royal Society of Chemistry 2013
 Table 1 Regression analysis and sample analysis data

Regression analysis for calibration line

Line equation y ¼ 2.026x + 5.547

Slope 2.026 T 0.06
Intercept 5.547 T 1.5
Correlation coefficient 0.992
Standard error of estimation 5.081

Sample analysis

Arsenic added Arsenic estimated

(ng) (ng) (%age error)

20 19.9 (0.50%)
36 35.9 (2.50%)
56 55.4 (2.85%)
80 78.9 (0.13%)
100 97.8 (2.20%)
Fig. 4 Color density as a function of reaction time. Field sample analysis

Arsenic found
Arsenic found by by conventional
Sample proposed method method

x T SD (mg L—1) RSD x T SD (mg L—1) RSD

Water from 2.2 T 0.06 0.296 2.3 T 0.10 0.505
tube well
(600–750ft depth)
Water from hand 8.9 T 0.21 0.261 8.7 T 0.15 0.195
pump
(70–120ft depth)
Canal water 12.3 T 0.15 0.138 12.4 T 0.15 0.138

spectrophotometric molybdenum blue method 23 to estimate

Fig. 5 Calibration line (color density signal as a function of arsenic
concentration in the reaction tube).
becomes too dark making it difficult for the software to distin-
guish between the individual colors.
Another benefit of the method which differs from the previous
arsenic was also practiced, and the results of these methods were
compared with those of the proposed method. The standard
deviation and RSD for 5 replicates for both the methods were
calculated and are reported in Table 1. It was startling to note that
the new method was more accurate even at very low concentra-
tions. The analysis of artificial samples and comparison with
standard methods showed that the present method was simple,
economical, accurate, portable, and user-friendly for the on-field
analysis of water samples to determine arsenic.
The interference of some common ions present in water (Na +,
K , SO4—2, Cl—, S—2) was also checked. It was found that these
+

methods is that it can be effectively employed to analyse samples
with large volumes. It was also found that using the assembly for
large volumes (100 mL) the detection limit of arsenic can be
further reduced (if desired) as for the same concentration of
arsenic in a large volume of solution a greater amount of arsenic
is present as compared to that present in a low volume assembly
(4 mL). For instance, 4 mL of 2 ppb solution contains 8 ng
arsenic, while 100 mL of 2 ppb contains 200 ng arsenic ions.
To corroborate the accuracy of the technique, 5 artificial
samples of different arsenic concentration were prepared and
analyzed. The concentration of arsenic was calculated by inter-
polating the software-calculated color density from the respective
calibration line. The experimental values were in close accordance
to the theoretical ones (Table 1). The proposed method was also
successfully applied to the field sample. Ground water and canal
water samples from different locations of Lahore, Pakistan were
chosen for the said purpose. Additionally, the conventional
ions produce no effect on the arsenic determination even at
a concentration 10 times the arsenic concentration except for S —2
ions. S—2 ions strongly interfere in the arsenic determination as
they are converted to H2S gas when they interact with acid. This
H2S gas can significantly affect the arsenic determination by
destroying the reagent paper. This interference was removed
using a lead acetate trap in the path of arsine.
Conclusion
The proposed method involves the determination of arsenic
without using sophisticated/expensive instruments. The modifi-
cations in the apparatus increase the precision and accuracy of
a well known Gutzeit method. The technique is easy to work and
can easily be applied as a kit method for on-field arsenic analysis.
It has an extremely low detection limit i.e. 1 ppb and a wide linear
range (2–20 ppb) which indicates its applicability at trace levels.

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 4, 242–246 | 245
The technique does not require special/specific sample prepara-
tion which is its positive impact. Furthermore the results are
reproducible. The technique has been successfully employed in
the analysis of several synthetic and field samples in order to
corroborate its efficiency.
References
1 F. N. Robertson, Environ. Geochem. Health, 1989, 11, 171–176.
2 J. H. T. Luong, E. Majid and K. B. Male, The Open Analytical
Chemistry Journal, 2007, 1, 7–14.
3 A. A. Nemodurk, The Analytical Chemistry of Arsenic.
Analiticheskaya Khimiya Mysh’yaka. Moscow: Nauka, 1976.
4 A. G. Howard and M. H. Arbab-Zavar, Analyst, 1981, 106, 213–220.
5 J. Aggett and G. Boyes, Analyst, 1989, 114, 1159–1161.
6 Y. Feng and J. Cao, Anal. Chim. Acta, 1994, 293, 211–218.
7 A. G. Howard and M. H. Arbab-Zavar, Analyst, 1980, 105, 338–343.
8 S. Kundu, S. K. Ghosh, M. Mandal, T. Pal and A. Pal, Talanta, 2002,
58, 935–942.
9 C. Pasha and B. Narayana, Bull. Environ. Contam. Toxicol., 2008, 81,
47–51.
246 | Anal. Methods, 2013, 4, 242–246
View publication stats
10 H. D. Revanasiddappa, B. P. Dayananda and T. N. K. Kumar,
Environ. Chem. Lett., 2007, 5, 151–155.
11 X. C. Le, W. R. Cullen and K. J. Reimer, Talanta, 1994, 41, 495–
502.
12 T. W. T. Rupasinghe, T. J. Cardwell, R. W. Cattrall and S. D. Kolev,
Anal. Chim. Acta, 2009, 652, 266–271.
13 A. Pazirandeh, A. H. Brati and G. Marageh, Appl. Radiat. Isot., 1998,
49, 753–759.
14 M. Kopanica and L. Novotny, Anal. Chim. Acta, 1998, 368, 211–218.
15 S. Jaya, T. P. Rao and G. P. Rao, Talanta, 1987, 34, 574–576.
16 A. S. Khan and A. Chow, Talanta, 1984, 31, 304–306.
17 W. Holak, Anal. Chem., 1969, 41, 1712.
18 I. Jaunakais, Int. Environ. Technol., 2002, 12, 2.
19 R. V. Abrazhee, A. D. Zorin, N. P. Savinova and Y. I. Sannikova, J.
Anal. Chem., 2002, 57, 280–283.
20 Hy. Almond, Anal. Chem., 1953, 25, 1766–7.
21 J. Anwar, Waheed-uz-Zaman, M. U. Shafique and M. Salman, Anal.
Lett., 2010, 43, 367–371.
22 U. Shafique, J. Anwar, M. Salman, Waheed-uz-Zaman, A. Dar,
R. Rehman, M. Azeem and S. Ameer, J. Sulfur Chem., 2011, 32, 151–
157.
23 Vogel, Textbook of Quantitative Inorganic Analysis, Ed-4th, 2004,
p. 732.
This journal is ª The Royal Society of Chemistry 2013