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Absorptive stripping voltammetry for cannabis detection
Department of Chemistry, Physical & Theoretical Chemistry Laboratory, Oxford University,

South Parks Road, Oxford OX1 3QZ, UK
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Chemistry Central Journal 2015, 9:41 doi:10.1186/s13065-015-0117-0
The electronic version of this article is the complete one and can be found online
at:http://journal.chemistrycentral.com/content/9/1/41
Received: 27 March 2015

Accepted: 19 June 2015
Published: 1 July 2015
2015 Nissim and Compton.
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited.
Abstract
Background
Given that 9 -tetrahydrocannabinol, the active constituent of cannabis, has been shown to
greatly reduce driving ability, thus being linked to many drug driving accidents, its reliable
detection is of great importance.
Results
An optimised carbon paste electrode, fabricated from graphite powder and mineral oil, is utilised
for the sensitive detection of 9 -tetrahydrocannabinol (THC) in both aqueous solutions of
pH 10.0 and in synthetic saliva solutions. Absorptive Stripping Voltammetry is exploited to
that effect and the paste is used to pre-concentrate the carbon paste electrode with the target
molecule. Practical limits of detection of 0.50 M and 0.10 M are determined for THC in
stationary and stirred aqueous borate buffer solutions, respectively. Theoretical limits of
detection are also calculated; values of 0.48 nM and 0.41 nM are determined for stationary and
stirred THC aqueous borate buffer solutions, respectively. THC concentrations as low as
0.50 M are detected in synthetic saliva solutions. The sensitivity of the sensor was
0.12 A M 1 , 0.84 A M 1 and 0.067 A M1 for the stationary buffer, the stirred buffer
and the saliva matrix, respectively.
Conclusions
Absorptive Stripping Voltammetry can be reliably applied to the detection of 9 -
tetrahydrocannabinol, after suitable optimisation of the assay. Usefully low practical limits of
detection can be achieved.
Keywords:
Absorptive stripping voltammetry; 9-tetrahydrocannabinol; Cannabis detection; THC detection;
Carbon paste electrode; Liquid-liquid interfaces
Background
9 -tetrahydrocannabinol (THC), the active constituent of cannabis, is known to reduce

psychomotor function and cognition thus greatly impairing driving ability [1], [2]. As would be
expected, reports indicate that the degree of impairment is related to the amount of THC present
in the body; as low as 0.95 M of THC in blood has been found to cause equivalent driving
impairment to an alcohol blood concentration of 11.5 mM, the legal limit in most European
countries [1]. At the same time, studies have also shown that cannabis is not only one of the most
commonly used illegal drugs [3], [4] but that it is also associated with many drug driving motor
vehicle accidents [5]. There is hence a great need for both accurate and low-concentration
detection as well as the development of methodologies suitable for road-side detection.
Cannabinoids are usually detected using gas chromatography [6][8] or high-performance liquid
chromatography, coupled to electrochemical detection [9][11]. However, given that
electrochemical sensors are known for their high reliability and speed of response, their low cost
and their compatibility for miniaturisation [12], they offer an attractive portable alternative[13]
[15]. Such sensors often rely on the indirect detection of THC, with sensing molecules including
Gibbs Reagent [13] and 4-aminophenol [14]. This is because the direct oxidation of the
hydroxyl group leads to the formation of radicals and radical cations which foul the electrode
surface [16][18]. This can significantly decrease the reproducibility of the obtained responses if
the sample is other than very dilute.
As previous studies have shown, carbon paste electrodes may, under open circuit conditions, be
loaded with an analyte of interest by being immersed in a solution containing that analyte[19]
[23]. The electrode can then be transferred into a blank solution, containing only buffer and
supporting electrolyte, in which the appropriate measurement can be carried out. It is likely that
the electrochemical reaction occurs at the triple phase boundary that exists at the carbon-oil
(binder)-water triple interface [22], as the analyte diffuses out of the paste. By using the bulk-
paste of an optimised carbon paste electrode to pre-concentrate THC and subsequently
stripping that target, the effect of fouling in the less dilute samples can be overcome [20]. The
pre-concentration of the target also ensures low limits of detection. This electroanalytical
approach has been termed absorptive stripping voltammetry, with concentration in the
electrode bulk [20], as opposed to adsorptive stripping voltammetry where the target is
allowed to adsorb on the electrode surface.
The aim of this paper is to apply absorptive striping voltammetry to the detection of low THC
concentrations in both aqueous solutions and in synthetic saliva, where the detection of THC will
rely on the direct oxidation of the molecule on an optimised carbon paste electrode. The value of
this approach has previously been indicated through work on the detection of different target
molecules such as superoxide [19], phenol [20] and vitamin K1 [21]. Here, the oxidation signal
of THC, observed at peak potentials of ca. +0.36 V [vs. saturated calomel electrode (SCE)], was
used as the detection signal, with the carbon paste being fabricated from graphite and mineral oil.
Experimental
Chemical reagents
All reagents were purchased from Aldrich (Gillingham, U.K.), at the highest grade available, and
were used as received, without any further purification. These were 9 -Tetrahydrocannabinol
(THC), dioctyl phthalate, mineral oil, graphite powder (particle diameter<20 m), synthetic
saliva, sodium hydroxide (NaOH), sodium tetraborate (Na 2 B 4 O 7 ) and potassium chloride
(KCl). All aqueous solutions were prepared daily, at 298 K, using deionised water of resistivity
of no less than 18.2 M cm (25 C, Millipore UHQ, Vivendi, U.K.) as the solvent and KCl
(0.1 M) as the supporting electrolyte. They were made at pH=10.0, achievable through the use
of appropriate NaOH/ Na 2 B 4 O 7 borate buffers (BBS solutions) and confirmed using a Hannah
pH 213 pH meter. Where experiments required the absence of oxygen [24], solutions were
deoxygenated using oxygen-free nitrogen (N 2 , BOC, Guildford, U.K.), in an air-tight
environment, for at least 30 min. The measurements themselves were carried out under a light
N 2 flow.
Equipment and experimental set-up
Square wave voltammetric measurements were recorded using a computer controlled Autolab
potentiostat (PGSTAT 101, EcoChemie, Utrecht, Netherlands), in a home-built Faraday cage. A
standard three-electrode configuration was used, with a carbon paste electrode (1.97 mm radius,
1.00 mm depth, made in-house) acting as the working electrode. A platinum wire (99.99 %
GoodFellow, Cambridge, U.K.) was utilised as the counter electrode and a Saturated Calomel
reference electrode (SCE, BAS Inc, Japan) completed the assembly. All experiments were
carried out in a thermostated water bath, at a temperature of 250.1 C.
Fabrication and characterisation of the carbon paste working electrodes
The carbon paste electrode holder was made from a copper rod of radius of 1.97 mm running
through a Teflon rod (for electrical contact), leaving a 1.00 mm deep cavity at the edge. Two
pastes were used, fabricated by mixing graphite powder with each liquid binder (dioctyl
phthalate and mineral oil). The graphite/dioctyl phthalate paste, fabricated by mixing 1.4 mL
dioctyl phthalate and 4.26 g graphite powder, has previously been characterised in aqueous
potassium ferricyanide [19]; the same ratio of pasting liquid to carbon powder was thus used to
make the graphite/mineral oil paste, unless otherwise stated.
Working electrode surface preparation
The surface of the carbon paste electrodes was renewed between each scan by cleaning the
holder and packing fresh paste. The errors in the calibration curves relate to separate electrode
preparations.
Results and discussion
The application of absorptive stripping voltammetry to the detection of THC is here discussed.
Given previous work [20] on the detection of phenols using this approach, a pre-concentration
time of 3 min was deemed adequate to equilibrate the paste with THC. An optimised paste
composition was used, where graphite powder and mineral oil were used for its fabrication; this
will be further discussed in Section 3.1. Practically useful limits of detection were thus achieved,
with the oxidation signal of THC, observed at peak potentials of ca. +0.35 V (vs. SCE), being
used as the detection signal.
Selecting a carbon paste electrode
The electrochemical oxidation of THC was first investigated using square wave voltammetry.
Two carbon paste electrodes, fabricated by mixing graphite powder with dioctyl phthalate or
mineral oil as described in Section 2.3, were used aiming to select the carbon paste electrode that
would ensure the highest THC uptake.
Each carbon paste electrode was immersed for 3 min, under open circuit conditions, in a
deoxygenated aqueous borate buffer solution of pH 10.0 that contained 7.0 80 M THC and
0.1 M KCl as the supporting electrolyte. Each paste electrode was then transferred to a
deoxygenated aqueous borate buffer solution of pH 10.0, which only contained 0.1 M KCl.
Oxidative square wave voltammetric scans were then run, between +0.25 V and+0.52 V (vs.
SCE) for the graphite/dioctyl phthalate paste electrode and between +0.20 V and +0.60 V (vs.
SCE) in the case of the graphite/mineral oil paste electrode. The frequency and step potential
used were 100 Hz and 1 mV, respectively, while the amplitude was set to 40 mV. Peaks due to
the oxidation of THC, as shown in Scheme 1[17], [25], [26], were seen at peak potentials of
+0.39 V and +0.37 V (vs. SCE) on the graphite/dioctyl phthalate and the graphite/mineral oil
pastes respectively; typical responses are depicted in Fig. 1. Scheme 1 assumes that THC
behaves like a typical phenol [17]; to the best of the authors knowledge there is no literature
reporting detection of further THC oxidation products.
Scheme 1
The oxidation of 9 -tetrahydrocannabinol (THC). THC has been assumed to behave as a typical
phenol [17], [25], [26]
Fig. 1. a Square wave voltammograms (frequency: 100 Hz, amplitude: 40 mV,

step potential: 1 mV) for the oxidation of THC, seen at +0.37 V (vs. SCE), on the
graphite/dioctyl phthalate paste electrode (black line) and the graphite/mineral oil paste electrode
(red line). The measurement was obtained in a deoxygenated BBS (0.1 M KCl, pH=10.0,
298 K), after immersing the electrode in identical stationary solutions that contained 80 M
THC, for 3 min. b Comparison of the peak currents obtained with the two carbon paste
electrodes (black squares: graphite/dioctyl phthalate paste, red circles: graphite/mineral oil
paste). The measurements were obtained in a deoxygenated BBS (0.1 M KCl, pH=10.0, 298 K),
after immersing the electrode in identicalstationary solutions that contained 7.0 80 M THC,
for 3 min. The errors relate to separate electrode preparations
As can be seen in Fig. 1a, b, the peak current observed with the graphite/mineral oil paste was
higher than that observed with the graphite/dioctyl phthalate paste, this reflecting the greater
ability of mineral oil to accumulate THC. As depicted in Fig. 1b, the current increased with
increasing analyte concentration, reflecting the increasing amount of THC transferring into the
paste. Only the graphite/mineral oil paste electrode was used for further experiments to achieve a
lower THC limit of detection.
Obtaining analytical limits of detection for THC
Detecting THC in a stationary vs. a stirred aqueous solution
Focusing on lower THC concentrations, the graphite/mineral oil paste was tested in aqueous
THC solutions of concentrations of 0.50 16 M. Analogous experiments as in the Section
above were thus carried out, where the pre-concentration time was increased to 5 min and where
the amount of mineral oil used was doubled; the amount of graphite powder was kept constant.
This was done to improve the uptake of THC and hence the sensitivity of the sensor.
The graphite/mineral oil carbon paste electrode was immersed in a stationary deoxygenated
aqueous borate buffer solution of pH 10.0 that contained a known amount of THC (between 0.50
and 16 M) and 0.1 M KCl as the supporting electrolyte. This was again done under open circuit
conditions. Oxidative square wave voltammetric scans were then run in a deoxygenated aqueous
borate buffer solution of pH 10.0, which only contained 0.1 M KCl, in the same potential range
(between +0.20 V and +0.60 V [vs. SCE]) and using the same values of 100 Hz, 1 mV and
40 mV for the frequency, step potential and amplitude, respectively, as in the Section above. The
same procedure was then repeated, stirring the source THC solutions to lower the limit of
detection of the sensor.
The results obtained from the stationary THC solution are presented in Fig. 2a, where peaks
corresponding to the oxidation of THC, as in Scheme 1, can be observed at a peak potential of
ca. +0.35 V (vs. SCE). As can be seen in Fig. 2b, the peak current shows a linear increase with
increasing THC concentration up to THC concentrations of 5.0 M; the peak height then levels
off and a plateau is reached when the THC concentration is further increased. Similarly, the
results obtained from the stirred THC solution can be seen in Fig. 3a, where the THC oxidation
peak is seen at a peak potential of +0.37 V (vs. SCE). As previously, the peak current increases
as the concentration of THC is increased, this time reaching a plateau at 4.0 M (Fig. 3b). The
errors in the calibration curves relate to separate electrode preparations.

step potential: 1 mV) for the oxidation of THC, seen at ca. +0.35 V (vs. SCE), on the
graphite/mineral oil paste electrode. The measurement was obtained in a deoxygenated BBS
(0.1 M KCl, pH=10.0, 298 K), after immersing the electrode in identical stationary solutions
that contained 0.50 16 M THC, for 5 min. b The increase of the peak current with increasing
THC concentration (black squares) with the correlation line through the linear range (red line,
R 2 =0.95). The lower practical limit of detection was determined as being 0.50 M, while the
slope of the calibration curve gave a value of 0.12 A M 1 for the sensitivity of the sensor. The
errors relate to separate electrode preparations

(0.1 M KCl, pH=10.0, 298 K), after immersing the electrode in identical stirred solutions that
contained 0.10 16 M THC, for 5 min. b The increase of the peak current with increasing THC
concentration (black squares) with the correlation line through the linear range (red line,
The above results are consistent with the fact that, as more THC is present in the source solution,
more THC will accumulate into the paste, resulting in higher peak currents being observed. The
fact that a plateau is reached indicates that, at some point, the detection is limited by the ability
of the paste to be loaded with more of the analyte. Comparing the THC concentration at which
the plateau is reached under stationary vs. stirred conditions, the reason it is reached at lower
THC concentrations when the solution is stirred is because the stirring replenishes the material
that is depleted near the electrode surface, as the analyte transfers into the paste. This leads to
more material accumulating into the paste at a given concentration.
Under stationary conditions, THC concentrations as low as 0.50 M could be practically
detected, while stirring the solution lowered the practical limit of detection to 0.10 M; again this
is due to the fact that stirring results in more material being present near the electrode surface
and hence available to accumulate into the carbon paste. Theoretical limits of detection (LODs)
were calculated by extrapolating the calibration curve and using the equation LOD =3/s, where
is the measured standard deviation of the signal in the absence of the target and s is the
sensitivity of the sensor [27]. These LOD values were determined as being 0.48 nM and 0.41 nM
for the stationary and stirred THC aqueous buffer solutions respectively. Limits of quantification
(LOQ) were also calculated by again extrapolating the calibration curve but using the equation
LOQ =10/s[27], where the variables are the same as in the LOD equation. The calculated
values were 1.61 nM and 1.38 nM for the stationary and stirred THC aqueous buffer solutions
respectively. Lastly, the slope of each calibration curve gave values for the sensitivity of the
sensor of 0.12 A M 1 and 0.84 A M 1 for the stationary and stirred THC aqueous buffer
solutions respectively.
Detecting THC in a stationary synthetic saliva solution
Focusing on the same THC concentration range as in Section 3.2.1, the graphite/mineral oil paste
was tested in THC synthetic saliva solutions of concentrations of 0.50 16 M. The same
conditions and carbon paste electrode as in the Section above were used.
The graphite/mineral oil carbon paste electrode was therefore immersed in a stationary
deoxygenated synthetic saliva/borate buffer solutions of pH=10.0 that contained a known
amount of THC (between 0.50 and 16 M) and 0.1 M KCl as the supporting electrolyte. This
was again done under open circuit conditions, using the same fixed 5 min pre-concentration time.
Oxidative square wave voltammetric scans were then run in a deoxygenated aqueous borate
buffer solution of pH 10.0, which only contained 0.1 M KCl, in the same potential range
40 mV for the frequency, step potential and amplitude, respectively, as in the Section above.
+0.37 V (vs. SCE). As can be seen in Fig. 4b, the peak current shows a linear increase with
increasing THC concentration up to THC concentrations of 7.0 M; a plateau is reached when
the THC concentration is further increased. The errors in the calibration curves relate to separate
electrode preparations.

graphite/mineral oil paste electrode. The measurement was obtained in a deoxygenated BBS (0.1
M KCl, pH=10.0, 298 K), after immersing the electrode in stationary deoxygenated synthetic
saliva/BBS solutions that contained 0.10 16 M THC, for 5 minutes. b The increase of the
peak current with increasing THC concentration (black squares) with the correlation line through
the linear range (red line, R 2 =0.99). The lower practical limit of detection was determined as
being 0.50 M, while the slope of the calibration curve gave a value of 0.067 A M 1 for the
sensitivity of the sensor. The errors relate to separate electrode preparations
The peak current increase was expected given that more and more THC will accumulate into the
paste if a higher amount of THC is present in the source solution. Again as expected, the uptake
is at some point limited by the paste, this resulting in the observed plateau. THC concentrations
as low as 0.50 M could be practically detected while the slope of the calibration curve gave a
value of 0.067 A M 1 for the sensitivity of the sensor. Theoretical LODs and LOQs were not
here calculated as the linear range was between 0.5 and 7.0 M.
Table 1 summarises the theoretical limits of detection for THC found in literature [11]
[13], [26],[28][30]. As expected, given the need for sensitive detection, sensitive methodologies
do exist, with limits of detection of as low as 3.2 nM having been reported [26]. However, the
LOD values for stirred and unstirred buffered matrices here calculated are much lower than those
tabulated. It is worth highlighting that the low practical limits of detection here determined
represent an observable signal.
Table 1. Summary of limits of detection of THC found in literature
Conclusions
Absorptive stripping voltammetry has been applied to the detection of THC in water and
saliva. An optimised carbon paste, made of graphite powder and mineral oil, was exploited in the
accumulation of THC, under open circuit conditions and using a 5 min pre-concentration time.
By testing the sensor in water and synthetic saliva samples of known THC concentrations,
analytical practical lower limits of detection (LODs) of 0.50 M and 0.10 M were obtained for
THC in stationary and stirred aqueous borate buffer solutions, respectively, and 0.50 M for
THC in stationary synthetic saliva solutions. Theoretical LOD values of 0.48 nM and 0.41 nM
were also calculated for the stationary and stirred buffer systems respectively. As Table 1 clearly
demonstrates, the theoretical limits of detection here determined are much lower than those
reported in the literature. Importantly, the practical limit of detection of 0.50 M determined in
synthetic saliva, and which corresponds to an observed signal, is comparable to theoretical
literature values, usually calculated. The limit of detection of 0.50 M is also practically useful
in terms of road side detection, this also clearly illustrating the value of absorptive stripping
voltammetry. This work opens up further studies into different ways of taking advantage of the
properties of carbon paste electrodes [31].
Competing interests
The authors declare that they have no competing interests.

Authors contributions
Both authors contributed equally to this work and have read and approved of the final
manuscript.
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Chemoenzymatic Kinetic resolution of (R)-malathion in aqueous media
Carlos A. Enrquez-Nez, Alejandro A. Camacho-Dvila, Vctor H. Ramos-
Snchez, Gerardo Zaragoza-Galn, Lourdes Ballinas-Casarrubias and David Chvez-
Flores*
*Corresponding author: David Chvez-Flores dchavezf@uach.mx
Author Affiliations
Facultad de Ciencias Qumicas, Universidad Autnoma de Chihuahua, Circuito No.1 Campus
Universitario, Chihuahua, Arboledas 31125, Chihuahua, Mxico

Chemistry Central Journal 2015, 9:46 doi:10.1186/s13065-015-0119-y
Received: 3 January 2015
Accepted: 10 July 2015
Published: 9 September 2015
2015 Enrquez-Nez et al.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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use, distribution, and reproduction in any medium, provided you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons license, and indicate if
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Formula display:
Abstract
Background
Malathion (R,S)-diethyl-2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate is a chiral
organophosphorus compound used widely as pesticide for suppression of harmful insects such as
mosquitoes. It is well known that in biological systems (R)-malathion is the active enantiomer,
therefore a sustainable approach could be the use of only the biologically active enantiomer. The
resolution of the commercial racemic mixture to obtain the pure active enantiomer combined
with a recycling of the undesired enantiomer through a racemization process could be an
attractive alternative to reduce the environmental impact of this pesticide. Thus, this work
evaluates the use of four commercially available lipases for enantioselective hydrolysis and
separation of malathion enantiomers from the commercial racemic mixture.
Results
Several lipases were methodologically assessed, considering parameters such as enzyme
concentration, temperature and reaction rates. Among them, Candida rugosa lipase exhibited the
best performance, in terms of enantioselectivity, E=185 (selective to the (S)-enantiomer). In this
way, the desired unreacted (R)-enantiomer was recovered in a 49.42 % yield with an
enantiomeric excess of 87 %. The monohydrolized (S)-enantiomer was recovered and racemized
in basic media, followed by esterification to obtain the racemic malathion, which was recycled.
In this way, an enantioenriched mixture of (R)-malathion was obtained with a conversion of
65.80 % considering the recycled (S)-enantiomer.
Conclusion
This work demonstrated the feasibility of exploiting Candida rugosa lipase to kinetically resolve
racemic malathion through an environmentally friendly recycling of the undesired (S)-
enantiomer.
Keywords:
Enantiomer; Enzymatic; Resolution; Malathion
Graphical abstract
Background
The importance of molecular chirality has been widely recognized in life sciences due to the
different activity of stereoisomers in biological systems. Chiral enantiomers have the same
chemical and physical properties in achiral environments, but they are often markedly different
in terms of their biological activity such as, toxicity and environmental fate in chiral
environments[1][3]. Organophosphorus pesticides (OP) are among the most important
chemicals used for protection against agricultural and household pests. It is estimated that OP are
worth nearly 40 % of the global market and they are expected to prevail in the near future. Chiral
pesticides currently account for about 33 % of the worldwide commercially available pesticides,
including some chiral OP. Among these, malathion (R,S)-[diethyl 2-
[(dimethoxyphosphorothioyl)sulfanyl]butanedioate] is one of the chiral OP extensively used for
insect and pests control on grains, fruits, nuts, cotton, and tobacco; which indeed is
commercialized as a racemic mixture: (R,S)-malathion [4]. It has been demonstrated that (R)-
malathion is the target-active enantiomer of this particular racemic mixture. As a matter of fact,
the (R)-enantiomer is 65x more toxic than the (S)-enantiomer [5],[6]. Therefore, the use of a
single enantiomer could result in a reduction of the amount applied to treat pests minimizing the
environmental impact and the cost of use. As the physical and chemical properties of
enantiomers are the same, the preparation of pure enantiomers is still a challenge, especially in
industrial processes. Generally, the industrial synthesis of chiral compounds generates the final
products as racemic mixtures, which are difficult to separate. As an alternative to obtain pure
enantiomers from racemic mixtures, the uses of enzymes is an attractive option.
Numerous biological processes are regulated by enzymes. In the last two decades, exploitation of
enzymes in synthetic organic chemistry increased significantly due to its potential to catalyze
reactions of specific substrates with high enantioselectivity and stereospecificity [7]. Lipases are
biocatalysts extracted in low yields from animals and plants. They can also be obtained in higher
yields by gene expression in an appropriate natural or recombinant microorganism. Lipases are
the most used enzymes in synthetic organic chemistry It has been demonstrated that they possess
a great versatility in catalysis of different reactions such as hydrolysis, esterification,
transesterification and aminolysis [8][11].
Several lipases have been used for the enantiomeric resolution of alpha substituted carboxylic
acids as ibuprofen, naproxen, ketoprofen, flurbiprofen, suprofen and other alpha and beta
substituted carboxylic acids [12][14]. Recently, we reported an enantioselective hydrolysis of
(S)-ibuprofen alkyl esters in aqueous media, also an enantioselective esterification in aqueous
media with different alcohol moieties using lipases as biocatalysts [14], [15].
Ideally, to obtain the pure (R)-malathion enantiomer, a lipase that selectively catalyzes the
transformation of the (S)-enantiomer should be used. Thus, the separation of unreacted (R)-
enantiomer could be performed easily by a simple extraction process. Some of the most widely
used lipases for the enantioselective hydrolysis of (S)-enantiomers include Candida
rugosa lipase,Candida antarctica lipase type B, Porcine pancreatic lipase and Mucor
javanicus lipase [12], [14],[16]. In this work, the above mentioned lipases were used as
biocatalysts for the enantioselective hydrolysis of (S)-malathion using commercially available
(R,S)-malathion as reagent. It is widely accepted that lipase-catalyzed reactions (hydrolysis,
esterification, alcoholysis, etc.) can be described by the ping-pong bi-bi mechanism, which
proceeds by a nucleophilic attack on the carbonyl group promoted by a serine, histidine and an
aspartate residues (also referred as catalytic triad), the resulting acyl enzyme intermediate
can forward react with a nucleophile, such as water, alcohols or amines, regenerating the enzyme
as shown in Fig. 1[17].
Fig. 1. The ping-pong bi-bi scheme mechanism

The equilibrium between hydrolysis and synthesis depends highly on the water content in the
reaction medium. Hydrolysis and ester synthesis are promoted by macro and micro aqueous
solvent systems, respectively [18]. The aim of this work was to find the best lipase-type enzyme
for enantioselective hydrolysis of (R,S)-malathion in aqueous media, in order to obtain the
unreacted (R)-enantiomer and the monohydrolized (S)-enantiomer. These two were separated
from each other and the latter was subsequently racemized in basic media and then esterified to
recover a racemic mixture, which was indeed recycled, thus improving the efficiency of the
overall process. The exploitation of pure or enriched (R)-enantiomer could improve the
efficiency of this pesticide by minimizing its dose and ultimately its environmental impact.
Enzyme assay
The hydrolytic activity of the four lipases was determined by a modified methodology previously
developed [19]. Sunflower oil was used as enzymatic substrate, where Candida rugosa lipase
showed the highest activity (31.8 U g 1 of biocatalyst), followed by porcine pancreatic lipase
(13.3 U g 1 of biocatalyst), Mucor javanicus lipase (5.6 U g 1 of biocatalyst) and Candida
antarcticalipase type B Novozym 435 (3.6 U g 1 of biocatalyst). It is important to emphasize
that the worst biocatalyst for this reaction was Candida antarctica lipase type B Novozym
435; despite being reported as a good biocatalyst for esterification and transesterification
reactions. However, in this context many aspects might influence its biocatalyst activity.
Perhaps, different enzyme sources (microorganism and mammalian vital organ) should naturally
be expected to exhibit structural differences, which influence strongly on biocatalysts properties
and activities, even in similar solvents [20]. In addition, the nature of the support and its polarity
can also affect the enzyme conformation, as well as the partition of substrates and products from
the enzyme environment, which might prevent the access of the substrate to the enzyme active
site. On this research, Candida antarctica lipase type B immobilized in a macroporous acrylic
resin was unable to catalyze the hydrolysis of sunflower oil and malathion, which is likely due to
the relative hydrophobic surface of the resin that difficult the interaction between the enzyme
active site and the substrate when the reaction occurs in an aqueous solvent [21].
Lipase-catalyzed enantioselective hydrolysis of racemic malathion
Based on the preliminary experiments described above, and in order to resolve the desired (R)-
enantiomer from the racemic mixture, an enantioselective hydrolysis of (R,S)-malathion into (S)-
malathion monocarboxylic acid and (R)-malathion was evaluated using Mucor
javanicus Lipase,Candida rugosa Lipase and Porcine pancreatic Lipase as biocatalysts
(Fig. 2). Candida antarcticaLipase type B (Novozym 435) was discarded because of its lack of
activity during the preliminary hydrolysis essay. We consider that lipases attack first the less
hindered ester group at position four farthest from the beta thioether substituent. The occurrence
of a dicarboxylic acid resulting from hydrolysis at the two ester groups was confirmed by chiral
HPLC chromatograms [22]. Only when the enzymatic reaction is left for more than 60 h,
degradation products were evidenced on the chiral HPLC chromatogram showing new signals at
retention times 9.673 min, 7.954 and 6.342 min [16].
Fig. 2. Lipase catalyzed enantiomeric resolution of racemic malathion
At the optimal reaction conditions (40 C, 250 rpm, 0.15:1 w:w ratio of enzyme:substrate and
phosphate buffer pH 7.2 as solvent) Candida rugosa lipase preferentially hydrolyze (S)-
malathion into (S)-malathion monocarboxylic acid allowing to recover (R)-malathion in a
satisfactory conversion yield (49.42 %) with a enantiomeric excess and enantioselectivity, of 87
and 185 respectively (Fig. 3). This was an obvious higher value than that obtained for Porcine
pancreatic lipase or Mucor javanicus lipase, (Table 1). However, as it was expected, their
performances were highly dependent on temperature and enzyme concentration. In fact, the best
temperature was 40 C for all reactions with enzyme concentrations 015 %, based on the
substrate weight. Thus, when (10 mmol, 3.3 g) of racemic substrate reacted, followed by
separation, extraction and evaporation at reduced pressure, in average 1.42 g of (S)-malathion
monocarboxylic acid were recovered. This amount is equivalent to 4.70 mmol a c =47.00 %.
This agrees in a 98.86 % with the conversion values obtained from the chiral HPLC
chromatograms areas, where the conversion determined was 47.54 %. The conversion and
enantioselectivity values for reactions catalyzed by Porcine pancreas and Mucor
javanicus lipases were very low, probably due to the poor dispersion of the enzyme on the
reaction media or by other factors already mentioned. Also the nonpolar solvent dependency
of Candida antarctica lipase type B was confirmed due to the low activity at conditions
investigated. Previous reports state that Candida antarctica lipase type B is denatured in aqueous
solvent above 40 C. Whereas in dry media, it can withstand temperatures above 100 C for
extended time [22][24]. As expected, the hydrolysis of racemic malathion in aqueous media
using Candida antarctica lipase type B did not occur within a range of temperature of 30 to
60 C. Considering the hydrophobicity of the macroporous acrylic resin and the aqueous solvent
on the reactions, we conclude that the substrate could not reach the enzyme active site.
Fig. 3. Chromatogram of the end of the enantioselective hydrolysis. (S)-

malathion monocarboxylic acid (tR=7.897 min), (R)-malathion (tR = 11.332 min), (S)-
malathion (tR =12.984 min)
Table 1. Lipase-catalyzed enantioselective hydrolysis of racemic malathion at optimal
conditions
Our temperature effect studies aided to establish that temperatures above 40 C leads to higher
conversion yields with a lower enantioselectivity. The optimal temperature for the kinetic
resolution of racemic malathion was determined to be 40 C with all lipases. Although several
studies of lipase kinetics have been carried out [21], the most common procedure is the use of the
pseudo-first order model [25]. Due to the absence of the correspondent UV signal in the chiral
HPLC chromatograms of the (R)-malathion monocarboxylic acid, it can be assumed that only
(S)-malathion was hydrolyzed by lipases. Under this hypothesis, using reaction data obtained
from the experiments, different kinetics were tested for the hydrolysis reactions of (R,S)-
malathion under the optimal conditions (40 C, barometric pressure and 250 rpm of agitation)
with the 3 active enzymes and all reactions fitted to the pseudo first-order kinetics [26].
Separation of enzymatic reaction products
The undesired (S)-malathion monocarboxylic acid was separated from the unreacted (R)-
malathion by extraction with a basic aqueous solution. It was noticed that the use of NaOH
solutions caused the racemization of both the (R)-malathion and the (S)-malathion monoester. To
prevent this, a 5 % aqueous solution of the less basic NaHCO3 was used. instead. The desired
(R)-malathion was then extracted with hexanes and evaporated at reduced pressure. The product
was analyzed by chiral HPLC (Fig. 4), polarimetry and NMR. The specific rotation of (R)-
malathion was determined to be in 96%ethanol, indicating an ee of at least

86 %, in good agreement with the literature [27], and matching closely with the ee =86.7
calculated using the chiral HPLC areas collected. These results are in contrast to mammalian
based hydrolyses where it has been demonstrated that (R)-malathion undergoes more rapid
degradation in the environment than the (S)-enantiomer [28][30].
Fig. 4. Chromatogram of the isolated (R)-malathion. (R)-malathion
(tR=12.984 min)
Racemization of (S)-malathion monocarboxylic acid
Once the (R)-malathion was isolated and characterized, the next goal was to racemize the
undesired (S)-enantiomer (Fig. 5). To carry out the racemization, aqueous Na2CO3 solution was
selected as base. It was found that when concentrations higher than 30 % of Na2CO3 were used,
the reaction took place at higher rates but resulting in a racemization and extensive hydrolysis of
the thiophosphonte ester bond. The optimal concentration was found to be at 10 % Na2CO3.
Thus, after the racemization, acidification and extraction with hexanes provided the racemized
product. Thus when 5 mmol (1.65 g) of (S)-malathion monocarboxylic were used for
racemization, 1.58 g of racemic malathion monocarboxylic acid (4.78 mmol) were recovered and
confirmed by chiral HPLC (Fig. 6). The final and desired product, (R)-malathion, was then was
characterized by chiral HPLC, polarimetry and FTIR which are in agreement with the literature.
Fig. 5. Chromatogram of the isolated (S)-malathion monocarboxylic acid. (S)-

malathion monocarboxylic acid (tR=7.897 min)
Fig. 6. Racemic malathion monocarboxylic acid. (S)-malathion monocarboxylic

acid (tR=7.897 min) and (R)-malathion monocarboxylic acid (tR=8.436 min)
Esterification of (R,S)-malathion monocarboxylic acid
Once the (R,S)-malathion monocarboxylic acid as recovered, it was submitted to an esterification
reaction to obtain the racemic malathion. This was carried out using excess of ethanol with a
catalytic amount of H 2 SO 4 in a Dean-Stark trap containing molecular sieves to remove the
formed water. In this way a conversion of 80.27 % yield was obtained. The obtaines racemic
malathion was characterized by HPLC, IR and NMR (Fig. 7).
Fig. 7. 1 H NMR spectrum of malathion
Experimental section
Too ensure the reproducibility and accuracy of the data each experiment was conducted in
triplicate. Results reported are the average values of the experiments.
Materials
Racemic malathion (R,S)-diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl] butanedioate was
isolated from inexpensive consumer pesticide Malathion 1000 donated by Velsimex S.A. de
C.V.Candida rugosa lipase, Candida antarctica lipase type B, Lipase from Porcine pancreas
and Mucor javanicus lipase were obtained from Sigma Aldrich Company. HPLC grade hexanes,
isopropanol, sodium phosphate monobasic and dibasic were purchased from MAESA Chemicals.
Cyclohexane, ethyl acetate and ethanol were purchased from FERMONT Company. All other
analytical grade reagents and solvents were obtained from commercially sources.
High performance liquid chromatography (chiral) analysis
In order to monitor the development of the enantioselective enzymatic hydrolysis, the product
separation and the acid catalyzed esterification reaction, high performance liquid
chromatography was performed with chirlacel OJ chiral column (Diacel Chemical Industries).
The HPLC instrument was equipped with a Dionex LPG-3400-D Quaternary Analytical Pump,
Dionex UltiMate 3000 Diode Array Detector, Dionex solvent degaser and Chromeleon CM-
PCS-1 Software. The mobile phase normally used was hexanes/isopropanol/trifluoroacetic acid
(95:4.9,0.1 % v/v/v). The UV detector wavelength was set to 254 nm, the flow rate was
1.0 mL/min and the temperatures of the column and injection compartments were 15 C. The
chromatographic signal peaks of racemic malathion were confirmed by comparing their retention
times (11.332 min (R)-malathion, 12.984 min for (S)-malathion and 7.897 min (S)-malathion
monocarboxylic acid) and UV spectra with the obtained with the reference standard. The optical
rotation was measured using an Atago POLAX 2 L semiautomatic Polarimeter at 22 C with a
sodium lamp at 589 nm using samples with concentrations of 1 g/100 mL in anhydrous ethanol.
Typically, about a gram of the mixture was separated and analyzed per run. A BCHI rotary
evaporator (Model R-210) was used to remove volatile solvents under reduced pressure. A
Perkin Elmer Fourier Transform Infrared Spectrometer Model IRGX with Attenuated Total
Reflection sampler was used for the characterization.
Isolation of (R,S)-malathion from commercially available pesticide formulation
Using 200 mL of cyclohexane as mobile phase in a 2.5 15 cm flash chromatography column
packed with 5 m silica gel as stationary phase, racemic malathion was isolated from a
commercially available malathion pesticide formulation. The mobile phase with the extracted
racemic malathion was evaporated at reduced pressure to isolate the racemic malathion. The
isolated material was analyzed by chiral HPLC, H 1 NMR and FTIR.
Lipase catalyzed enantioselective hydrolysis of malathion
In a typical reaction, 3.30 g (10 mmol) of racemic malathion, 40 mL of 0.1 M sodium phosphate
buffer pH 7.02, 0.1650.66 g of lipase and 0.5 g of Celite 577 fine for dispersion of lipase
particles were added in to a 100 mL dry baffled-flat-bottom flask. The reactions were stirred at
300 rpm and 40 C. The mixture was analyzed by chiral HPLC before adding lipase. By taking
1 mL samples and using extraction with hexanes, the reactions were monitored for at least 48 h
by chiral HPLC and then stopped and centrifuged at 4500 rpm for 6 min for the enzyme recovery
by decanting the reaction solution. The remaining oil was weighed and saved for further
separation and analysis.
Enantioselectivity value (E-value) measurements

The value of enantioselectivity (E ) was calculated from the enantiomeric excess of the substrate
(ee ) and the conversion degree (c ) according to the equations 1 and 2 described by Chen et
al.[23].
(1)
(2)
Isolation of (R)-malathion and racemization of (S)-malathion monocarboxylic acid

In order to isolate the desired product (R)-malathion and to avoid its racemization, a weak base
was used to produce the (S)-malathion sodium monocarboxylate. About 40 mL of the decanted
reaction solution was extracted three times with 40 mL of 5 % v/w NaHCO 3 aqueous solution.
The desired (R)-malathion was isolated by removing the solvent by evaporation at reduced
pressure.
Racemization of (S)-malathion monocarboxylic acid
In a 100 mL round bottom flask the undesired subproduct (S)-malathion monocarboxylic acid
1.51 g (5 mmol) was mixed with 40 mL of aqueous Ca 2 CO 3 at different concentrations 5, 10,
15, 20, 25 and 30 % w/w. Then the mixture was stirred for 60 min. In order to monitor the
racemization reactions, 1 mL of sample was removed from the reaction solution to slowly be
acidified with 3 % HCl w/v to induce the formation of the corresponding malathion
monocarboxylic acid. Once the racemization was achieved, the solution was acidified and the
racemic mixture was extracted by triplicated with hexanes and evaporated at reduced pressure to
be analyzed by chiral HPLC determining the residual weight and enantiomeric excess.
Esterification of (R,S)-malathion monocarboxylic acid
Typically in a 100 mL baffled-flat-bottom flask 1.51 g (5 mmol) of (R,S)-malathion
monocarboxylic acid, 5 mL of ethanol and 30 mL of solvent (anhydrous ethanol, acetone,
dioxane and acetonitrile) and 1 mL of concentrated sulfuric acid were added. The mixture was
refluxed and stirred for 8 h using a Dean Stark apparatus and molecular sieves to remove water,
the undesired byproduct. The reaction mixture was extracted by triplicated with 30 mL of
cyclohexane. The combined organic layers were dried with magnesium sulfate and evaporated at
reduced pressure to give (R,S)-malathion. The compound matched perfectly with the physical
characteristics of malathion, an oily compound with garlic odor and yellow color. The isolated
malathion was analyzed by Chiral HPLC and the optical rotation matching satisfactorily with the
standard of reference.
Conclusion
Comparison of commercially available lipases as biocatalysts for the enantioselective hydrolysis

of (R,S)-malathion was performed. the obtained results suggested that Candida rugosa lipase was
the best biocatalyst of the lipases studied, showing conversion yields up to 49.47 %, with
acceptable enantioselectivity of E=185 to the S enantiomer. It was found that temperature of
40 C and a weight ratio of 0.15:10 for substrate:enzyme were optimal for all reactions. The
monohydrolyzed (S)-malathion, was successfully racemized and esterified obtaining, (R,S)-
malathion for further recycling. In conclusion, this work demonstrated the feasibility of
exploiting Candida rugosa lipase to kinetically resolve racemic malathion through an
environmentally friendly recovery of the undesired (S)-enantiomer. This methodology could also
be applied for the resolution of other OPs such as Phentoate, which also contains a chiral group
and it is indeed a pesticide widely used worldwide.
Competing interests
CE-N carried out the experimental work. DC-F is the corresponding author and wrote most of
the manuscript. AC-D carried out the spectroscopic characterization, LB-C and GZ-G carried out
the chromatographic analysis. VR-S contributed to data analysis and interpretation. All authors
read, approved and contributed equally to the manuscript.
Acknowledgments
We gratefully acknowledge the financial support of the Mexican Department of Education (F-
PROMEP-38/Rev-03 SEP-23-005), the Department of Chemistry of the Autonomous University
of Chihuahua and VELSIMEX SA de CV.
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A solid-phase extraction method using Transcarpathian clinoptilolite for preconcentration
of trace amounts of terbium in water samples
Volodymyr O. Vasylechko12*, Galyna V. Gryshchouk1, Victor P. Zakordonskiy1, Olga
Vyviurska1 and Andriy V. Pashuk3
*Corresponding author: Volodymyr O Vasylechko vasylechko@ukr.net
Author Affiliations
1
Faculty of Chemistry, Ivan Franko National University of Lviv, 6 Kyryla and Mefodiya St.,
Lviv, 79005, Ukraine
2
Department of Chemistry and Physics, Lviv Academy of Commerce, 9 Samchuka St., Lviv,
79011, Ukraine
3
Department of Environmental Safety and Nature Protection, Lviv Polytechnic National
University, 12 Bandera St., Lviv, 79013, Ukraine
Chemistry Central Journal 2015, 9:45 doi:10.1186/s13065-015-0118-z
Received: 5 January 2015
Published: 27 August 2015
2015 Vasylechko et al.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
changes were made.
Formula display:
Abstract
Background
In spite of the fact that terbium is one of the rarest elements in the Earths crust, it is frequently
used for the production of high technological materials. At the result, an effective combination of
sample preparation procedure and detection method for terbium ions in different matrices is
highly required. The solid-phase extraction procedure with natural Transcarpathian clinoptilolite
thermally activated at 350 C was used to preconcentrate trace amounts of terbium ions in
aqueous solutions for a final spectrophotometric determination with arsenazo III.
Results
Thermogravimetric investigations confirmed the existence of relations between changes that
appeared during dehydratation of calcined zeolite and its sorption affinity. Since the maximum of
sorption capacity towards terbium was observed at pH 8.25, a borate buffer medium
(2.510 4 ) was used to maintain ionic force and solution acidity. Terbium was quantitatively
removed from the solid-phase extraction column with a 1.0 M solution of sodium chloride
(pH 2.5). The linearity of the proposed method was evaluated in the range of 2.5-
200 ngmL 1 with detection limit 0.75 ngmL1 .
Conclusions
Due to acceptable recoveries (93.3102.0 %) and RSD values (67.1) from spiked tap water, the
developed method can be successfully applied for the determination of trace amounts of terbium
ions in the presence of major components of water.
Keywords:
Preconcentration; Terbium; Solid-phase extraction; Clinoptilolite
Graphical abstract
Background
Terbium belongs to the rarest elements, participating with 1.110 4 % in the composition of the
Earths crust [1], and this is only 1.0 % among all lanthanides including yttrium. At the same
time terbium compounds have been widely applied for luminophores, magnetic and laser
materials. Furthermore, this lanthanide was detected in sea and mineral waters [2] as well as in
some wines at the microlevel. For example, the content of terbium and other rare earth elements
was used for authentication of Hungarian wines [3]. In general, the determination of
microelements from real samples and wastewaters requires a proper sample preparation
procedure, including such steps as preconcentration, separation and isolation from natural objects
and process liquors. In this case, ion exchange and extraction chromatography were found to be
effective and fast for the separation of trace amounts of rare earth elements [4], [5]. At the same
time, the solid-phase extraction (SPE) has also become quite popular sample preparation
procedure for trace analysis[5][9], allows to reduce solvent consumption due to a simplified
procedure of the solvent removal. Furthermore, SPE could be easily combined with the selective
and high sensitive methods of the analysis, e.g. atomic absorption spectroscopy [9], [10] and
inductively coupled plasma spectroscopy [5][7]. A variety of sorbents such as modified high
dispersed silica [5], [11], [12], activated carbon [5], [13], [14], polyurethane foams [15], [16],
polymeric resins [9], [17], carbosilicon [18], synthetic zeolites [19] are commonly used for solid
phase extraction. In recent years, the popularity of natural zeolites has increased for SPE
applications [6], [10], [20][29], because of a number of advantages over the other sorbents. For
example, these natural aluminosilicate minerals contain pores and cavities with strictly defined
size and shape, and it provides very effective concentration and separation of organic and
inorganic compounds. In addition, zeolites have mechanical strength, good stability in aggressive
medium and under thermal treatment, ability to sorb the trace amounts of analytes, high sorption
capacity and selectivity, possibility of easy modification and regeneration of the sorbent, low
cost and accessibility. The sorption properties of Transcarpathian clinoptilolite towards terbium
ions were described in our previous work [30]. The aim of this study is to complete these
investigations and to develop a simple sample preparation procedure for the spectrophotometric
detection of trace amounts of terbium ions in aqueous solutions.
Experimental
Reagents and apparatus

All used reagents were of analytical grade. Standard aqueous solutions of terbium(III) nitrate
(1.0 mgmL 1 ) were prepared by dissolution of metallic terbium (99.9 % purity) in nitric acid
(1:1). The following solutions were also used for the experiments: a 0.05 % solution of
sulfarsazene in a 0.05 M borax solution, a 0.05 % aqueous solution of arsenazo III, a 1 %
ascorbic acid solution, a formic buffer solution (60 mL of formic acid and 28 g of NaOH are
dissolved in 1.0 L of water), a 1 M sodium chloride solution, a 5 % sulphosalicylic acid solution,
a 0.2 M EDTA solution, a 0.1 M aqueous solutions of NaOH and Na 2 B 4 O 7 , and a borate
buffer solution with pH 8.25 (595 mL of 0.05 M Na 2 B 4 O 7 diluted to 1.0 L with 0.1 M HCl).
Clinoptilolite samples with 8590 % of the main component content were taken from the deposit
near the village Sokirnytsia in the Ukrainian Transcarpathian region. The specific surface area
measured with water sorption was found to be 59 m 2 g 1[31]. The chemical composition of
Transcarpathian clinoptilolite is (in %): SiO 2 , 67.29; TiO 2 , 0.26; Al 2 O 3 , 12.32; Fe 2 O 3 ,
1.26; FeO, 0.25; MgO, 0.99; CaO, 3.01; Na 2 O, 0.66; K 2 O, 2.76; H 2 O, 10.90 [32]. The
thermal heating of Transcarpathian clinoptilolite was carried out for 2.5 h in the oven. The
results of the X-ray analysis of the calcined Transcarpathian clinoptilolite were described in
details in our previous study [33]. Spectrophotometric determination was performed on a HACH
DR/4000 V spectrophotometer. X-ray fluorescence investigations were carried out on an Expert
3 L multi element rapid response analyzer (INAM, Ukraine) with semiconducting PIN-detector
(AMPTEK, USA) on thermoelectric cooling. A low-power X-ray tube was operated at 45 kV
(current 0.1 mA, output 4.5 W). In this case, 2 mL of the sample was placed into the cuvette, and
Tb L line radiation was measured for 285 s. The PaulikPaulikErdey Q1500D (MOM,
Hungary) system was employed for thermogravimetric analysis.
Adsorption measurements
The sorption properties of clinoptilolite were studied under dynamic conditions using a
peristaltic pump, and the procedure was described in details in our previous paper [34]. The
metal solutions were passed through a sorption cartridge filled with 0.6 g of sorbent at a flow rate
of 3 mLmin 1. A passing moment of terbium(III) ions (LDL, 100 ngmL 1 ) was detected
visually and/or spectrophotometrically at 540 nm with sulfarsazene as a chromogenic reagent,
and additionally confirmed by X-ray fluorescence.
An acidified solution of sodium sulphate and mineral acids solutions were used for desorption of
Tb(III) ions from clinoptilolite. In this case 15 mL of eluent was passed through the sorption
cartridge at a flow rate of 1 mLmin 1 . The obtained eluate was collected into a volumetric
glass flask and made up to 25 mL using double-distilled water.Since selectivity of determination
of Tb(III) ions with sulfarsazene was insufficient for the analysis of desorption filtrates, a content
of Tb(III) ions was found spectrophotometrically with arsenazo III as a reagent [35], [36].
Interference of Fe(III), Al(III), Ca(II) and Mn(II) ions was eliminated by the addition of ascorbic
and sulphosalicylic acids, EDTA and Seignette salt to the system. The adsorption and desorption
studies were carried out at a temperature of 201 C.
Method of spectrophotometric determination with arsenazo III
5 mL of an 1 % ascorbic acid solution, 4 mL of a 4 % sulphosalicylic acid solution, 5 mL of a
0.2 M EDTA solution, 3 mL of a 5 % Seignette salt solution were mixed with 25 mL of the
analysed solution (pH~1). After 2 min, a mixture of 1 mL of a formic buffer solution (pH 3.5)
and 4 mL of a 0.05 % arsenazo III solution was diluted with double-distilled water to
approximately 40 mL and regulated to pH 2.60.1 with a 0.1 M NaOH. The final volume of the
solution was made up to 50 mL with double-distilled water, and the absorbance was measured
spectophotometrically at 650 nm against the blank solution which contains all the reagents
except for terbium(III) ions. The quantitative determination of terbium(III) ions was carried out
in the final volume of the solution (in concentration range of 0.12 gmL 1 ).
Thermogravimetric analysis
The analysis was performed under air conditions with the heating rate 10 Cmin 1 from 50 to
900 C with Al 2 O 3 as a standard material. Clinoptilolite samples (grain size 0.200.31 mm)
weighting 500510 mg were placed in corundum crucibles. The sorbent was preliminary heated
in a drying oven or a muffle oven during 2.5 h and stored in a desiccator above a saturated
solution of H 2 SO 4 .
As can be seen from the Fig. 1, the sorption capacity of Transcarpathian clinoptilolite towards
terbium(III) ions considerably depends on the pH of analyte solution and previous thermal
treatment of the used zeolite. The most effective sorption was observed in the weak alkaline
solutions (pH 8.25), where according to our previous studies [30], terbium(III) ions exist in three
cation forms of Tb 3+ (~25 %), TbOH 2+ (~50 %) and Tb(OH) 2+ (~25 %). In order to maintain
pH and improve the accuracy of the investigations, a borate buffer was added to terbium
solution. Consequently, it was found that the same amounts of terbium(III) ions were
concentrated from the solution adjusted to pH 8.25 with sodium hydroxide at once and from the
solution which was firstly neutralized, then buffered to pH 8.25. Moreover, the use of the buffer
solution provides a possibility to keep constant ionic strenght, and minimize the negative
influence of different admixtures on the effectiveness of terbium preconcentration.
Fig. 1. Dependence of clinoptilolite sorption capacity of terbium(III) ions on a
pH value of the aqueous solution and thermal treatment carried out in the range from 20 to
700 C (pH 8.25) (concentration of Tb(III) 1 gmL 1 ; pH 8.25; a flow rate 3 mLmin 1 ;
time of heat treatment 2.5 h)
As to the thermal treatment of Transcarpathian clinoptilolite, a minimum at 250 C and a
maximum at 350 C of sorption capacity for terbium were observed over a narrow temperature
range (Fig. 1). These observations confirmed the connection between zeolite structural changes
during dehydration processes and its sorption abilities [23][31], [33], [34], [37]. At the same
time, only partial rehydration of zeolite could be observed in distilled water, e.g. water content of
Transcarpathian clinoptilolite was reduced by 18 wt. % after thermal treatment at 500 C. As a
result, the sorption effectiveness of thermally activated zeolite towards trace element ions was
not further diminished in aqueous solutions.
The results of our previous investigations [30] showed that efficiency of exchangeable cations
and specific surface value of clinoptilolite change after thermal treatment of the sorbent.
Similarly, specific surface area and sorption capacity of thermally treated clinoptilolite samples
depend on the pretreatment temperature. Conseqently, an additional thermogravimetric
experiment was carried out to investigate the thermal desorption of water from the clinoptilolite
surface. Figure 2illustrates TG-and DTG-curves of natural clinoptilolite and clinoptilolite
thermally activated at 500 C. It appears that TG-and DTG-curves have almost identical shapes.
TG-curve entirely represents physical and chemical transformations of the sample during
continuous heating. The observed plateau at 600700 has an S-form which is typical for
minerals. At the same time, TG-curve suggests the influence of previous thermal treatment of the
sorbent on its adsorption capacity for water. These results could be confirmed by DTG-curves
(Fig. 3) and data of the overall water loss at 900 C for Transcarpathian clinoptilolite heated at
different temperatures (Table 1). For instance, the samples thermally activated at 200250 C
were characterized with a minimum of water loss (6.1 %). Relatively high amounts of water loss
(9.49.8 %) were received for the clinoptilolite previously calcined at the temperature range of
300500 C. In this case, the reverse dependence between water loss amounts and temperature of
thermal treatment was received. Nevertheless, previous heating of the sample at 700 C again
caused a sharp decrease in clinoptilolite water loss.
Fig. 2. TG and DTG-curves of natural clinoptilolite (1, 1*) and clinoptilolite

heated at 500 (2, 2*)
Fig. 3. DTG-curves of Transcarpathian clinoptilolite previously heated at

different temperatures
Table 1. Overall water loss at 900 C for clinoptilolite samples previously heated at different
temperatures
Consequently, it could be proposed that, the temperature of the zeolite thermal pretreatment
influences on a rate of adsorption equilibrium rather than on its sorption capacity of water at
200250 C. This suggestion could be confirmed by the fact that weight losses were 9.9 % for
the sorbent previously heated at 200 and 250 C and then kept for 72 h in air with relative
humidity of 70 %.
These effects appeared from decelerated process of rehydration, since changes of clinoptilolite
porous structure were still reversible at 200250 C. On the contrast, an irreversible deep
amorphization of Transcarpathian clinoptilolite occured at 700 C [33]. In general, DTG-curve is
a differential curve of the sample weight change and describes the dependence of the weight loss
rate from the temperature. For the studied clinoptilolite samples, DTG-curves were asymmetrical
and some characteristic points were identified (Fig. 3). A broad DTG-maximum observed at low
temperatures indicates the intensive thermal desorption of water. It should be noted that
maximum rates of water loss were observed for all studied samples in the temperature range
from 110 to 130 (140) C, independently from previous thermal treatment conditions.
Furthermore, this dissymmetry of the DTG-maximum could be caused by overlapping between a
few quasielementary maximums, which suggests at least two types of molecular water physically
bonded to the clinoptilolite surface. In this temperature range, the water loss did not significantly
varied with the temperature of the sorbent preparation (6.37.0 %), except for the sample
calcinated at 700 C (4.4 %). An isokinetic region (indicated by arrows on Fig. 3) was observed
on DTG-curves at the temperature of 440560 C for all samples, apart from the sorbent
previously heated at 700 C.
Moreover, an undefined DTG-maximum was found for the clinoptilolite previously calcined at
250 C. It is known that a deep dehydroxylation of silica occurs in this temperature range [38],
and practically all OH-groups appear to be isolated at temperature over 400 C. Consequently,
the fully dehydroxylated surface is covered with oxygen atoms because of recombination of two
hydroxyl groups and release of water molecules. This process demands reorganization of the
surface atoms and also should be activated. Since surface dehydroxylation of the Transcarpathian
clinoptilolite considerably diminishes the amount of surface OH-groups responsible for the
sorption of trace elements ions, the terbium sorption effectiveness of clinoptilolite heated above
400 C was significantly decreased (Fig. 1).
It has been reported [39] that water molecules in the hydrated zeolite could form the cyclic
hexamers with oxygen atoms of the sorbent framework. Due to these hydrogen bonds, water
molecules did not contain free OH-groups. Moreover, such cyclic hexamers prevent sorption of
large cationic aqua and hydroxo complexes of metal ions. Desorption of ligand water molecules
caused a simultaneous destruction of hydrogen bonds and cyclic hexamers at the temperature
above 200 C. As a result, a number of free OH-groups considerably increased due to water
molecules of the broken hexamer, which were still bonded to the zeolite framework. Since the
surface OH-groups are mainly active sorption centers towards trace elements ions, a noticeable
improvement of sorption properties of the clinoptilolite heated in the temperature range of 250
350 C towards terbium ions (Fig. 1) was connected with the increased number of surface
hydroxyl groups provided by water molecules and surface silanol (SiOH) groups.
Permissible multiple contents ( ion / Tb(III) ) of ions common in waters and wastewaters, that do
not change the maximum sorption capacity of clinoptilolite towards terbium ions, are led in
Table 2. Increasing of the admixture concentration beyond a define value leads to the reduction
in sorption effectiveness of the zeolite.
Table 2. Tolerance limits of some ions for terbium(III) sorption from aqueous solution of
Transcarpathian clinoptilolite (concentration of Tb(III) 1 gmL 1 ; pH 8.25)
A 7 M solution of HNO 3 and a 1 M solution of NaCl acidified with a hydrochloric acid to
pH 2.5 were preferred as desorbents for terbium(III) ions, because each of them provided almost
full recovery of lanthanide (Table 3). As a result, the developed solid phase extraction procedure
could be applied for the spectrophotometric determination of trace amounts of terbium(III) ions
in aqueous samples.
Table 3. Desorption effectiveness of terbium(III) ions from clinoptilolite a
Sample preconcentration procedure
The sorbent was grounded in a ball mill to 0.200.31 mm size and washed with distilled water.
After drying at room temperature, the clinoptilolite sample was heated in the oven at 350 C for
2.5 h and then stored in a desiccator. 0.52 L water sample was acidified with nitric acid to a pH
value approximately 1 and heated on a sand bath for 1 h. After the filtration through the dense
paper filter, the pH of the water was adjusted to 7 with a sodium hydroxide solution, and then a
borate buffer was added to maintain pH 8.25. The final concentration of a borate buffer in the
sample solution was 2.510 4 . The obtained solution passed through SPE cartridge filled
with 0.6 g of the prepared sorbent at a flow rate of 3 mLmin 1 . After the loading of the
sample, the cartridge was washed with 50 mL of double-distilled water at the same flow rate.
Whereas, terbium(III) ions were desorbed with 15 mL of a 1 M sodium chloride solution
acidified with a hydrochloric acid to pH 2.5 at a flow rate 1 mLmin 1 . 5 mL of double-
distilled water was added to the eluate, and the pH was adjusted to 1 with hydrochloric acid. The
obtained solution was made up to 25 mL volume with double-distilled water and thoroughly
mixed. Terbium(III) content in the solution was determined spectrophotometrically with
arsenazo III as an indicator. This procedure is described in detail in the experimental part.
Overall, the proposed method for determination of Tb(III) ions had a linearity range from 2.5 to
200 ngmL 1 . The detection limit was found to be 0.75 ngmL 1 , and this parameter was
calculated using the following equation:
[Math Processing Error]
where Sb is a standard deviation of blank and m is a slope of the calibration curve. The sample
preparation method was tested on a model solution with the composition similar to natural water.
As can be seen from Table 4, the components of waters do not have considerable influence on
the determination of trace amounts of terbium(III) ions. The analyte recoveries from spiked tap
water were above 93 %.
Table 4. Determination of terbium(III) ions in the synthetic water sample with the composition
similar to natural surface waters after ions preconcentration with clinoptilolite (n=3, P=0.95)
Conclusions
A solid-phase extraction procedure was developed for spectrophotometric determination of trace

amounts of terbium(III) ions in aqueous solution. Transcarpathian clinoptilolite heated at 350 C
for 2.5 h was applied as a SPE sorbent. Maximum sorption ability towards terbium(III) ions was
observed after thermal activation of the clinoptilolite. The results of thermogravimetric
investigations indicated a relationship between changes of zeolite structure during dehydration
processes and its sorption abilities. It was shown that previous thermal treatment of clinoptilolite
has an influence on its sorption capacity and the overall water loss, which suggests only partial
reversibility of zeolite rehydration. Due to this fact, thermally activated samples of clinoptilolite
maintain their sorption abilities towards trace elements ions, especially terbium(III) ions, in
aqueous solutions.
The same sorption values were obtained for terbium(III) solutions with pH 8.25 regulated with
either a borate buffer or sodium hydroxide. A buffer solution maintains a pH value, which
permits to improve metrological characteristics of the measurements. Moreover, in this case the
constant ionic strenght minimizes influence of other water components on the sorption process of
Tb(III) ions. The developed method offers a possibility to concentrate trace amounts of
terbium(III) ions in the presence of water components. Permissible multiple contents of
competing ions for terbium(III) ions sorption were the following: 2500 (Cl ), 2000 (NH 4+ ,
NO 3 ), 1500 (Na + , K +), 1000 (SO 42 ), 300 (Mg 2+ ), 50 (Ca 2+ , Zn 2+ ). A 1.0 M sodium
chloride solution acidified with hydrochloric acid to pH 2.5 was used for quantitative desorption
of terbium(III) ions., An enrichment factor of 130 was obtained under the optimum conditions. A
wide range of linearity (2.5200 ngmL 1 ) with detection limit of 0.75 ng. mL 1 was achieved.
The developed procedure was applied for the determination of terbium(III) ions in technological
solutions, where recoveries and RSD values were 93.3103.0 % and 1.67.1, respectively.
Competing interests
VOV conceived of the ideas of the project, carried out SPE investigation and coordinated the
project. VPZ was responsible for the thermogravimetric analysis, AVP performed X-ray
fluorescence determination of terbium. OV defined the influence of borax solution on the
effectiveness of the SPE extraction, and GVG determined the optimum SPE conditions fo
terbium concentration. All authors contributed to the preparation of the manuscript, read and
approved the final version.
Acknowledgement
This work was partially funded by the Ministry of Education and Science of Ukraine.
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Promising upshot of silver nanoparticles primed fromGracilaria crassa against bacterial
pathogens
V Lavakumar1, K Masilamani2, V Ravichandiran3, N Venkateshan4, D V R Saigopal5, C
K Ashok Kumar1 and C Sowmya6*
*Corresponding author: C Sowmya drsowmyariper@gmail.com
Author Affiliations
1
Department of Pharmaceutical Biotechnology, Sree Vidhyanikethan College of Pharmacy,
A.Rangampet, Tirupati 517102, AP, India
2
Faculty of Technology, University Malaysia Pahang, Lebuhraya Tun Razak, Gambang,
Kuantan, 26300, Pahang Darul Makmur, Malaysia
3
National Institute of Pharmaceutical Education and Research, Kolkata 700032, WB, India
4
Department of Pharmaceutical Chemistry, Sankarlingam Bhuvaneswari College of
Pharmacy, Sivakasi 626130, TN, India
5
Department of Virology, S.V. University, Tirupati 517502, AP, India
6
Department of Pharmaceutics, Raghavendra Institute of Pharmaceutical Education and
Research, Anantapuram 515721, AP, India
2015 Lavakumar et al.

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Abstract
Background
The study on newer antimicrobial agent from metal based nano materials has augmented in
recent years for the management of multidrug resistance microorganisms. In our present
investigation, we synthesized silver nanoparticles (AgNPs) from red algae, Gracilaria crassa as
beginning material which effectively condensed the silver ions to silver nanoparticles with less
price tag and no risk.
Methods
Silver nanoparticles were prepared by simple reaction of 1 mM AgNO 3 with G. crassa extracts
at room temperature. The fabricated AgNPs were subjected for characterization and screened
against various microorganisms for antibacterial activity.
Results
UVVis spectroscopy (200800 nm), XRD, FESEM and EDAX, were performed for AgNPs.
UVVis spectroscopy demonstrated the absorption edge at 443 nm and EDAX pattern is purely
due to the particle size and face centered cubic (fcc) symmetry of nanoparticles. Average size
lays at 122.7 nm and zeta potential was found to be 34.9 mV. The antibacterial outcome of
synthesized AgNPs (at the dose of 20 and 40 g/ml) was evaluated against Escherichia
coli, Proteus mirabilis,Bacillus subtilis and Pseudomonas aeruginosa. The mechanism of
synthesized AgNPs bactericidal bustle is discussed in terms of interaction with the cell
membrane of bacteria. The activity was found to be sky-scraping in a dose dependent manner.
Conclusion
Thus, environmental friendly, cost effective, non hazardous stable nanoparticles were prepared
by green synthesis using red algae, G. crassa. Synthesized G. crassa AgNPs were in acceptable
size and shape. Further, it elicits better bactericidal activity against microorganism. This will
assure the out put of superior antibacterial formulation for near future.
Graphical Abstract
Keywords:
Green synthesis; Red algae; Gracilaria crassa ; Silver nanoparticles; Antibacterial activity
Graphical abstract
Background
From ancient, handling of microbial infection is an exigent task for microbiologists. Countless
drugs have been found to be successful, unfortunately, it leads to mount of drug resistance
against particular pathogens with an outlook of stern issues in concern with public wellbeing [1].
Technical community is animatedly trying to expand groundbreaking concepts in drug delivery
by challenging the new microbial agent with superior mode of action by its effectual target on
the cell membrane or neither on intracellular targets [2], [3]. In 21st century, nanotechnology has
become inevitable, not because of only claim and also by the way of synthesis. Two way
synthesis like physical and chemical methods have several sizeable challenges like technically
protracted, expensive and ecological hazardous defects [4]. The current art of exploration in
research is heading towards green synthesis of high-yield, squat in cost, non-hazardous and eco-
friendly metallic nanoparticles by plants and microorganisms [5], [6]. Due to hefty surface area,
high reactivity and surface plasmon resonance, these nanoparticles were tailored for definite
application by scheming into unambiguous size, shape and morphology leading to exhibit its
broad spectrum of activity against multi drug resistance microorganisms [7][10]. A quest for an
environmentally sustainable synthesis process has led to a few biomimetic approaches like
applying natal principles in the formation of nanoparticles. Among several, bioreduction is the
prime and widely practiced functional method in synthesizing the nano materials[11], [12].
Noble metal nanoparticles such as silver, gold, which are geared up by plant extracts, algae,
bacteria and fungi are broadly applied in drug delivery systems [13], electronics [14], [15],
biosensors [16], cancer therapeutics and antimicrobial agents [17][19]. For three decades,
exploration of marine algae has been far above the ground for the search of new and effective
natural origin medicines, because it posses elevated quantity of concealed bioactive essence.
Several of such compounds, including carbohydrates, alkaloids, steroids, phenols, saponins and
flavonoids, etc. [20][22]. These multifaceted compounds exhibit a wide range of industrial and
biotechnological applications [23]. An extent, these bio-molecules play decisive role in reduction
of metal ions and generate the stable eco friendly nanoparticles. Further, AgNPs fetches
superior antibacterial activity by interacting with thiol clusters present in bacterial cell by cliping
its replication. Literature strappingly supports that these Ag + ions unyoke the respiratory chains
and collapse the proton motive forces across the cytoplasmic membrane of
bacteria [24]. Gracilaria crassa (G. crassa), a well-known red algae, having potential secondary
metabolites [25]. In harmony to the above information, the present study was intended to prepare
and typify silver nanoparticles from G. crassa, further to explore its antimicrobial activities
against highly resistance microbial inhabitants.
Methods
Materials
Silver nitrate (AgNO 3 ; Mol. Wt: 169.87; Prod. No: 27462) of analytical grade (AR) was
purchased from Fisher scientific, Mumbai, India. The nutrient agar medium was purchased from
Hi Media (Mumbai, India). All other chemicals used were analytical grade. Microorganisms
such as Escherichia coli (MTCC 443), Proteus mirabilis (MTCC 442), Bacillus subtilis (MTCC
441) andPseudomonas aeruginosa (MTCC 424) were obtained from Microbial Type Culture and
Collection, Pune, India.
Seaweed collection and extraction
Gracilaria crassa was collected along the coast of Mandapam (91658.9N 791853.6E),
Rameswaram, Tamilnadu, India. The freshly collected algal material was rinsed with seawater
followed by deionized water to get rid of extra impurities. The samples were kept in shade for
15 days drying. The algal material was ground to powder and uphold stockpile by placing at
4C for further studies. The G. crassa extract was prepared in a conical flask by taking 2.5 g in
100 mL of deionized water. It was heated for 45 min at 50C and positioned in an orbital shaker
for 24 h in order to conquer the maximum extraction of compounds. The Extracts were filtered
through whatman No. 1 filter paper and stored in refrigerator for further studies [26].
Synthesis of silver nanoparticles
AgNPs were synthesized by adopting the method proposed by Sathishkumar et al. with simple
modification [27]. 5 ml of algal extract was added in to 95 ml of 1 mM aqueous silver nitrate
solution, in 250 ml conical flasks and kept at 30C in a shaker for overnight to facilitate absolute
reduction. The samples were monitored periodically for its color intensity to confirm the
formation of AgNPs.
Characterization of silver nanoparticles
UVVis spectral analysis
The reduction in pure silver ions was recorded by measuring the UVVis spectrum of the
synthesized AgNPs of G. crassa at room temperature with a Perkin Elmer Lambda 25 UVVis
spectrometer at the wavelength of 200800 nm [28].
Particle size and zeta potential studies
Particle size and zeta potential experiments for G. crassa AgNPs were carried out by using a
HORIBA Instruments (Singapore) Pvt Ltd, Singapore.
Powder x-ray diffraction (XRD) analysis
The silver nanoparticles were alienated by repeated centrifugation at 12,000 rpm for 10 min
followed by redispersion of AgNPs into deionized water for three times. The supernatant was
transferred in microwave for drying. X-ray diffraction (XRD) measurement of the AgNPs was
carried out using Rigaku smart lab instrument (Japan), function at voltage of 40 kV, 30 mA
current with Cu K1 radiations.
FESEM-EDAX studies
After careful UVVis spectroscopical analysis of synthesized nanoparticles, diameter of
nanoparticles were further confirmed by Field emission scanning electron microscopyenergy
dispersive X-ray analysis (FESEMEDAX) by using SUPRA 55-CARL ZEISS, Germany.
Antibacterial assay
The antibacterial evaluation of AgNPs was conceded by using different experimental pathogens
like Escherichia coli (MTCC 443), Proteus mirabilis (MTCC 442), Bacillus subtilis (MTCC 441)
andPseudomonas aeruginosa (MTCC 424) maintained by department of Pharmaceutical
Biotechnology, SVCP, Tirupati, India. Prior to experimentation, untainted cultures was
subcultured into nutrient media. Nutrient agar plates were prepared, seeded and pierced with
20 and 40 g/ml of AgNPs. Streptomycin sulphate (20 g/ml) was used as standard [29].
UVVis spectral analysis

The formation of AgNPs was defined by color transformation [30] from pale yellow to dark
brown (Fig. 1). The color change is due to the excitation of the surface plasmon resonance (SPR)
(Fig. 2) which elicits max of 443 nm.
Fig. 1. Formation of silver nanoparticles by green synthesis. AgNPs were

formed by the reduction of silver ions by Gracilaria crassa. This figure illustrates the various
stages of formation of AgNPs; a pure algal extracts (pale yellow); b at 0 min, on immediate
addition of 1 mM solution of Silver nitrate (no reaction); c after 15 min (slight reduction); d after
30 min (moderate reduction); e after 12 h of addition of 1 mM solution of Silver nitrate
(Complete reduction).
Fig. 2. UV-Vis absorption maxima of silver nanoparticles. The data is based on

the presence of absorbance peak of AgNPs solution at the wavelength range of 400450 nm.
The absorption maxima were found to be 434 nm.
Particle size and zeta potential
Particle size determination of synthesized AgNPs was revealed underneath by intensity. Laser
diffractions exposed by obtained AgNPs were in polydisperse concoction with average size of
122.7 nm (Fig. 3). The zeta potential endows stability of nanoparticles and surface charge. The
zeta potential was found to be 34.9 mV. Earlier reports strongly supports, when the zeta
potential value positions between 30 and 50 mV, it specifies good stability of nano
particles [31]. The high negative value (Fig. 4) substantiates the repulsion between the particles
and thereby increases the stability of the AgNPs.
Fig. 3. Particles size distribution of AgNPs prepared from G. crassaextracts.

Average particle size of synthesized AgNPs ranges from 60 to 200 nm
Fig. 4. Zeta potential illustration of AgNPs. Zeta potential distribution of

synthesized AgNPs prepared from G. crassa extracts.
X-ray diffraction (XRD) analysis
The XRD pattern of powder sample of G. crassa AgNPs exhibited peaks at 38, 44, 64 and
77. Four Braggs reflections corresponding to (111), (200), (220) and (311) planes of the fcc
crystal structures of metallic silver (JCPDS No. 89-3722) are interpreted from the XRD (Fig. 5).
The orientation (111) is more predominant since it shows high intense. Broadening of the
diffraction crest disclose the formation of pure crystalline silver [32].
Fig. 5. X-ray diffraction analysis of bio synthesized silver nanoparticles using

marine red algae extracts of G. crassa corresponds four Braggs reflections planes of the metallic
silver nanoparticles.
FESEM and energy dispersive X-ray analysis
Field emission scanning electron microscopy investigation was further confirmed the size of
silver nanoparticles synthesized from G. crassa. The size (diameter) of the nanoparticles ranges
between 60 and 200 nm and the shapes were spherical and some are irregular (Fig. 6). The
outcome of FESEM reports were overlapped with earlier reports [33], [34]. The energy
dispersive X-ray analysis (EDAX) depicts strong indication in the silver region, which
authenticate formation of silver nanoparticles (Fig. 7). The optical absorption peak at 3 kV,
which attributed to metallic silver nanocrystallites owing to surface plasmon
resonance [35], [36].
Fig. 6. Morphological images of synthesized AgNPs by using FESEM. This

figure shows individual nanoparticles in clusters, spherical and some with irregular texture.
Fig. 7. Energy dispersive X-ray analysis of silver nanoparticles. EDAX profile of
AgNPs shows higher percentage of silver signal.
Antibacterial assay
Antibacterial activity by agar well diffusion technique was recorded after 24 h incubation of
culture plates. The AgNPs demonstrate excellent antibacterial activity against all tested
microorganisms (Fig. 8). AgNPs showed high spectrum of activity against E. coli and P.
mirabilis at the concentrations of 20 and 40 g/ml when compared with standard (Fig. 9). The
significant zone of inhibition was exerted due to effect of AgNPs on biochemical process of the
bacterial cell by interacting thiol and amino groups of proteins and nucleic acids of cell
wall [37][39]. Further, this could lead to interaction between nanoparticles and
microorganism which results in triggering the discharge of highly reactive oxygen species
(ROS), mostly hydroxyl radicals and singlet oxygen[40][42]. This augments the deposition of
nanoparticles on the bacterial cell surface results large accumulation of silver nanoparticles
causing disruption of cellular functions.
Fig. 8. Antibacterial activity of AgNPs. Zone of inhibition of silver

nanoparticles against aEscherichia coli, bProteus mirabilis, cBacillus subtilis, dPseudomonas
aeruginosa; [T 1- (20 g/ml of AgNPs) and T 2(40 g/ml of AgNPs); Standard (Std-
Streptomycin sulphate 20 g/ml)].
Fig. 9. Sensitivity prototypes of silver nanoparticles against various microbial

pathogens.
Conclusion
In summary, the bio-reduction of aqueous silver ions to silver nanoparticles (AgNPs) was
successfully done using marine red algae, G. crassa in trouble-free, economy and ecofriendly
manner. The average size of silver nanoparticles was found to be 122.7 nm with high stability of
34.9 mV. Further characterization by UVVis spectroscopy, FESEM, EDAX confirm
formation nanoparticles which are virtually spherical in shape. XRD divulges fcc structural
confirmation. The proved antibacterial potential will lend a hand to develop a powerful
antibacterial formulation in near future as biomedical remedies. Hence, authors robustly propose
this green synthesis of nanoparticles can be extended to the wide range of applications.
Abbreviations
AgNPs: silver nanoparticles
FESEM: field emission scanning electron microscopy
EDAX: energy dispersive analysis of X-rays
XRD: X-ray diffraction
Fcc: face centered cubic
Cu: copper
mV: millivolts
SPR: surface plasmon resonance
kV: kilovolts
mM: millimolar
nm: nanometer
ROS: reactive oxygen species
MTCC: microbial type culture and collection
LKV and SC are designed, performed and wrote the manuscript. MK contributed for
instrumentation. RV & VN helped in interpretation of FESEM and XRD. AK & SG were helped
in screening antimicrobial activity. All authors read and approved the final manuscript.
Acknowledgements
The authors are grateful to T.N.K.V. Prasad, Institute of Frontier Technology, Regional
Agricultural Research Station, Acharya N.G. Ranga Agricultural University, Tirupati for helping
us to carry out particle size analysis. The authors also thankful to Center for nanotechnology,
Sathyabhama University, Chennai for carrying out FESEM and XRD studies. The authors
extended thanks to Sree Vidyanikethan College of Pharmacy, A.Rangampet, Tirupathi for
providing facilities to carry out this work.
Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.
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Absorptive stripping voltammetry for cannabis detection
Rita Nissim and Richard G Compton*
*Corresponding author: Richard G Comptonrichard.compton@chem.ox.ac.uk
Author Affiliations
Department of Chemistry, Physical & Theoretical Chemistry Laboratory, Oxford University,
South Parks Road, Oxford OX1 3QZ, UK

Accepted: 19 June 2015
Published: 1 July 2015
2015 Nissim and Compton.

This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited.
Abstract
Background
Given that 9 -tetrahydrocannabinol, the active constituent of cannabis, has been shown to
greatly reduce driving ability, thus being linked to many drug driving accidents, its reliable
detection is of great importance.
Results
An optimised carbon paste electrode, fabricated from graphite powder and mineral oil, is utilised
for the sensitive detection of 9 -tetrahydrocannabinol (THC) in both aqueous solutions of
pH 10.0 and in synthetic saliva solutions. Absorptive Stripping Voltammetry is exploited to
that effect and the paste is used to pre-concentrate the carbon paste electrode with the target
molecule. Practical limits of detection of 0.50 M and 0.10 M are determined for THC in
stationary and stirred aqueous borate buffer solutions, respectively. Theoretical limits of
detection are also calculated; values of 0.48 nM and 0.41 nM are determined for stationary and
stirred THC aqueous borate buffer solutions, respectively. THC concentrations as low as
0.50 M are detected in synthetic saliva solutions. The sensitivity of the sensor was
0.12 A M 1 , 0.84 A M 1 and 0.067 A M1 for the stationary buffer, the stirred buffer
and the saliva matrix, respectively.
Conclusions
Absorptive Stripping Voltammetry can be reliably applied to the detection of 9 -
tetrahydrocannabinol, after suitable optimisation of the assay. Usefully low practical limits of
detection can be achieved.
Keywords:
Absorptive stripping voltammetry; 9-tetrahydrocannabinol; Cannabis detection; THC detection;
Carbon paste electrode; Liquid-liquid interfaces
Background
9 -tetrahydrocannabinol (THC), the active constituent of cannabis, is known to reduce

psychomotor function and cognition thus greatly impairing driving ability [1], [2]. As would be
expected, reports indicate that the degree of impairment is related to the amount of THC present
in the body; as low as 0.95 M of THC in blood has been found to cause equivalent driving
impairment to an alcohol blood concentration of 11.5 mM, the legal limit in most European
countries [1]. At the same time, studies have also shown that cannabis is not only one of the most
commonly used illegal drugs [3], [4] but that it is also associated with many drug driving motor
vehicle accidents [5]. There is hence a great need for both accurate and low-concentration
detection as well as the development of methodologies suitable for road-side detection.
Cannabinoids are usually detected using gas chromatography [6][8] or high-performance liquid
chromatography, coupled to electrochemical detection [9][11]. However, given that
electrochemical sensors are known for their high reliability and speed of response, their low cost
and their compatibility for miniaturisation [12], they offer an attractive portable alternative[13]
[15]. Such sensors often rely on the indirect detection of THC, with sensing molecules including
Gibbs Reagent [13] and 4-aminophenol [14]. This is because the direct oxidation of the
hydroxyl group leads to the formation of radicals and radical cations which foul the electrode
surface [16][18]. This can significantly decrease the reproducibility of the obtained responses if
the sample is other than very dilute.
As previous studies have shown, carbon paste electrodes may, under open circuit conditions, be
loaded with an analyte of interest by being immersed in a solution containing that analyte[19]
[23]. The electrode can then be transferred into a blank solution, containing only buffer and
supporting electrolyte, in which the appropriate measurement can be carried out. It is likely that
the electrochemical reaction occurs at the triple phase boundary that exists at the carbon-oil
(binder)-water triple interface [22], as the analyte diffuses out of the paste. By using the bulk-
paste of an optimised carbon paste electrode to pre-concentrate THC and subsequently
stripping that target, the effect of fouling in the less dilute samples can be overcome [20]. The
pre-concentration of the target also ensures low limits of detection. This electroanalytical
approach has been termed absorptive stripping voltammetry, with concentration in the
electrode bulk [20], as opposed to adsorptive stripping voltammetry where the target is
allowed to adsorb on the electrode surface.
The aim of this paper is to apply absorptive striping voltammetry to the detection of low THC
concentrations in both aqueous solutions and in synthetic saliva, where the detection of THC will
rely on the direct oxidation of the molecule on an optimised carbon paste electrode. The value of
this approach has previously been indicated through work on the detection of different target
molecules such as superoxide [19], phenol [20] and vitamin K1 [21]. Here, the oxidation signal
of THC, observed at peak potentials of ca. +0.36 V [vs. saturated calomel electrode (SCE)], was
used as the detection signal, with the carbon paste being fabricated from graphite and mineral oil.
Experimental
Chemical reagents
All reagents were purchased from Aldrich (Gillingham, U.K.), at the highest grade available, and
were used as received, without any further purification. These were 9 -Tetrahydrocannabinol
(THC), dioctyl phthalate, mineral oil, graphite powder (particle diameter<20 m), synthetic
saliva, sodium hydroxide (NaOH), sodium tetraborate (Na 2 B 4 O 7 ) and potassium chloride
(KCl). All aqueous solutions were prepared daily, at 298 K, using deionised water of resistivity
of no less than 18.2 M cm (25 C, Millipore UHQ, Vivendi, U.K.) as the solvent and KCl
(0.1 M) as the supporting electrolyte. They were made at pH=10.0, achievable through the use
of appropriate NaOH/ Na 2 B 4 O 7 borate buffers (BBS solutions) and confirmed using a Hannah
pH 213 pH meter. Where experiments required the absence of oxygen [24], solutions were
deoxygenated using oxygen-free nitrogen (N 2 , BOC, Guildford, U.K.), in an air-tight
environment, for at least 30 min. The measurements themselves were carried out under a light
N 2 flow.
Equipment and experimental set-up
Square wave voltammetric measurements were recorded using a computer controlled Autolab
potentiostat (PGSTAT 101, EcoChemie, Utrecht, Netherlands), in a home-built Faraday cage. A
standard three-electrode configuration was used, with a carbon paste electrode (1.97 mm radius,
1.00 mm depth, made in-house) acting as the working electrode. A platinum wire (99.99 %
GoodFellow, Cambridge, U.K.) was utilised as the counter electrode and a Saturated Calomel
reference electrode (SCE, BAS Inc, Japan) completed the assembly. All experiments were
carried out in a thermostated water bath, at a temperature of 250.1 C.
Fabrication and characterisation of the carbon paste working electrodes

The carbon paste electrode holder was made from a copper rod of radius of 1.97 mm running
through a Teflon rod (for electrical contact), leaving a 1.00 mm deep cavity at the edge. Two
pastes were used, fabricated by mixing graphite powder with each liquid binder (dioctyl
phthalate and mineral oil). The graphite/dioctyl phthalate paste, fabricated by mixing 1.4 mL
dioctyl phthalate and 4.26 g graphite powder, has previously been characterised in aqueous
potassium ferricyanide [19]; the same ratio of pasting liquid to carbon powder was thus used to
make the graphite/mineral oil paste, unless otherwise stated.
Working electrode surface preparation
The surface of the carbon paste electrodes was renewed between each scan by cleaning the
holder and packing fresh paste. The errors in the calibration curves relate to separate electrode
preparations.
The application of absorptive stripping voltammetry to the detection of THC is here discussed.
Given previous work [20] on the detection of phenols using this approach, a pre-concentration
time of 3 min was deemed adequate to equilibrate the paste with THC. An optimised paste
composition was used, where graphite powder and mineral oil were used for its fabrication; this
will be further discussed in Section 3.1. Practically useful limits of detection were thus achieved,
with the oxidation signal of THC, observed at peak potentials of ca. +0.35 V (vs. SCE), being
used as the detection signal.
Selecting a carbon paste electrode
The electrochemical oxidation of THC was first investigated using square wave voltammetry.
Two carbon paste electrodes, fabricated by mixing graphite powder with dioctyl phthalate or
mineral oil as described in Section 2.3, were used aiming to select the carbon paste electrode that
would ensure the highest THC uptake.
Each carbon paste electrode was immersed for 3 min, under open circuit conditions, in a
deoxygenated aqueous borate buffer solution of pH 10.0 that contained 7.0 80 M THC and
0.1 M KCl as the supporting electrolyte. Each paste electrode was then transferred to a
deoxygenated aqueous borate buffer solution of pH 10.0, which only contained 0.1 M KCl.
Oxidative square wave voltammetric scans were then run, between +0.25 V and+0.52 V (vs.
SCE) for the graphite/dioctyl phthalate paste electrode and between +0.20 V and +0.60 V (vs.
SCE) in the case of the graphite/mineral oil paste electrode. The frequency and step potential
used were 100 Hz and 1 mV, respectively, while the amplitude was set to 40 mV. Peaks due to
the oxidation of THC, as shown in Scheme 1[17], [25], [26], were seen at peak potentials of
+0.39 V and +0.37 V (vs. SCE) on the graphite/dioctyl phthalate and the graphite/mineral oil
pastes respectively; typical responses are depicted in Fig. 1. Scheme 1 assumes that THC
behaves like a typical phenol [17]; to the best of the authors knowledge there is no literature
reporting detection of further THC oxidation products.
Scheme 1
The oxidation of 9 -tetrahydrocannabinol (THC). THC has been assumed to behave as a typical
phenol [17], [25], [26]

step potential: 1 mV) for the oxidation of THC, seen at +0.37 V (vs. SCE), on the
graphite/dioctyl phthalate paste electrode (black line) and the graphite/mineral oil paste electrode
(red line). The measurement was obtained in a deoxygenated BBS (0.1 M KCl, pH=10.0,
298 K), after immersing the electrode in identical stationary solutions that contained 80 M
THC, for 3 min. b Comparison of the peak currents obtained with the two carbon paste
electrodes (black squares: graphite/dioctyl phthalate paste, red circles: graphite/mineral oil
paste). The measurements were obtained in a deoxygenated BBS (0.1 M KCl, pH=10.0, 298 K),
after immersing the electrode in identicalstationary solutions that contained 7.0 80 M THC,
for 3 min. The errors relate to separate electrode preparations
As can be seen in Fig. 1a, b, the peak current observed with the graphite/mineral oil paste was
higher than that observed with the graphite/dioctyl phthalate paste, this reflecting the greater
ability of mineral oil to accumulate THC. As depicted in Fig. 1b, the current increased with
increasing analyte concentration, reflecting the increasing amount of THC transferring into the
paste. Only the graphite/mineral oil paste electrode was used for further experiments to achieve a
lower THC limit of detection.
Obtaining analytical limits of detection for THC
Detecting THC in a stationary vs. a stirred aqueous solution
Focusing on lower THC concentrations, the graphite/mineral oil paste was tested in aqueous
THC solutions of concentrations of 0.50 16 M. Analogous experiments as in the Section
above were thus carried out, where the pre-concentration time was increased to 5 min and where
the amount of mineral oil used was doubled; the amount of graphite powder was kept constant.
This was done to improve the uptake of THC and hence the sensitivity of the sensor.
The graphite/mineral oil carbon paste electrode was immersed in a stationary deoxygenated
aqueous borate buffer solution of pH 10.0 that contained a known amount of THC (between 0.50
and 16 M) and 0.1 M KCl as the supporting electrolyte. This was again done under open circuit
conditions. Oxidative square wave voltammetric scans were then run in a deoxygenated aqueous
borate buffer solution of pH 10.0, which only contained 0.1 M KCl, in the same potential range
40 mV for the frequency, step potential and amplitude, respectively, as in the Section above. The
same procedure was then repeated, stirring the source THC solutions to lower the limit of
detection of the sensor.
ca. +0.35 V (vs. SCE). As can be seen in Fig. 2b, the peak current shows a linear increase with
increasing THC concentration up to THC concentrations of 5.0 M; the peak height then levels
off and a plateau is reached when the THC concentration is further increased. Similarly, the
results obtained from the stirred THC solution can be seen in Fig. 3a, where the THC oxidation
peak is seen at a peak potential of +0.37 V (vs. SCE). As previously, the peak current increases
as the concentration of THC is increased, this time reaching a plateau at 4.0 M (Fig. 3b). The
errors in the calibration curves relate to separate electrode preparations.

(0.1 M KCl, pH=10.0, 298 K), after immersing the electrode in identical stationary solutions
that contained 0.50 16 M THC, for 5 min. b The increase of the peak current with increasing
THC concentration (black squares) with the correlation line through the linear range (red line,

(0.1 M KCl, pH=10.0, 298 K), after immersing the electrode in identical stirred solutions that
contained 0.10 16 M THC, for 5 min. b The increase of the peak current with increasing THC
concentration (black squares) with the correlation line through the linear range (red line,
The above results are consistent with the fact that, as more THC is present in the source solution,
more THC will accumulate into the paste, resulting in higher peak currents being observed. The
fact that a plateau is reached indicates that, at some point, the detection is limited by the ability
of the paste to be loaded with more of the analyte. Comparing the THC concentration at which
the plateau is reached under stationary vs. stirred conditions, the reason it is reached at lower
THC concentrations when the solution is stirred is because the stirring replenishes the material
that is depleted near the electrode surface, as the analyte transfers into the paste. This leads to
more material accumulating into the paste at a given concentration.
Under stationary conditions, THC concentrations as low as 0.50 M could be practically

detected, while stirring the solution lowered the practical limit of detection to 0.10 M; again this
is due to the fact that stirring results in more material being present near the electrode surface
and hence available to accumulate into the carbon paste. Theoretical limits of detection (LODs)
were calculated by extrapolating the calibration curve and using the equation LOD =3/s, where
is the measured standard deviation of the signal in the absence of the target and s is the
sensitivity of the sensor [27]. These LOD values were determined as being 0.48 nM and 0.41 nM
for the stationary and stirred THC aqueous buffer solutions respectively. Limits of quantification
(LOQ) were also calculated by again extrapolating the calibration curve but using the equation
LOQ =10/s[27], where the variables are the same as in the LOD equation. The calculated
values were 1.61 nM and 1.38 nM for the stationary and stirred THC aqueous buffer solutions
respectively. Lastly, the slope of each calibration curve gave values for the sensitivity of the
sensor of 0.12 A M 1 and 0.84 A M 1 for the stationary and stirred THC aqueous buffer
solutions respectively.
Detecting THC in a stationary synthetic saliva solution
Focusing on the same THC concentration range as in Section 3.2.1, the graphite/mineral oil paste
was tested in THC synthetic saliva solutions of concentrations of 0.50 16 M. The same
conditions and carbon paste electrode as in the Section above were used.
The graphite/mineral oil carbon paste electrode was therefore immersed in a stationary
deoxygenated synthetic saliva/borate buffer solutions of pH=10.0 that contained a known
amount of THC (between 0.50 and 16 M) and 0.1 M KCl as the supporting electrolyte. This
was again done under open circuit conditions, using the same fixed 5 min pre-concentration time.
Oxidative square wave voltammetric scans were then run in a deoxygenated aqueous borate
buffer solution of pH 10.0, which only contained 0.1 M KCl, in the same potential range
40 mV for the frequency, step potential and amplitude, respectively, as in the Section above.
+0.37 V (vs. SCE). As can be seen in Fig. 4b, the peak current shows a linear increase with
increasing THC concentration up to THC concentrations of 7.0 M; a plateau is reached when
the THC concentration is further increased. The errors in the calibration curves relate to separate
electrode preparations.

graphite/mineral oil paste electrode. The measurement was obtained in a deoxygenated BBS (0.1
M KCl, pH=10.0, 298 K), after immersing the electrode in stationary deoxygenated synthetic
saliva/BBS solutions that contained 0.10 16 M THC, for 5 minutes. b The increase of the
peak current with increasing THC concentration (black squares) with the correlation line through
the linear range (red line, R 2 =0.99). The lower practical limit of detection was determined as
being 0.50 M, while the slope of the calibration curve gave a value of 0.067 A M 1 for the
sensitivity of the sensor. The errors relate to separate electrode preparations
The peak current increase was expected given that more and more THC will accumulate into the
paste if a higher amount of THC is present in the source solution. Again as expected, the uptake
is at some point limited by the paste, this resulting in the observed plateau. THC concentrations
as low as 0.50 M could be practically detected while the slope of the calibration curve gave a
value of 0.067 A M 1 for the sensitivity of the sensor. Theoretical LODs and LOQs were not
here calculated as the linear range was between 0.5 and 7.0 M.
Table 1 summarises the theoretical limits of detection for THC found in literature [11]
[13], [26],[28][30]. As expected, given the need for sensitive detection, sensitive methodologies
do exist, with limits of detection of as low as 3.2 nM having been reported [26]. However, the
LOD values for stirred and unstirred buffered matrices here calculated are much lower than those
tabulated. It is worth highlighting that the low practical limits of detection here determined
represent an observable signal.
Table 1. Summary of limits of detection of THC found in literature
Conclusions
Absorptive stripping voltammetry has been applied to the detection of THC in water and
saliva. An optimised carbon paste, made of graphite powder and mineral oil, was exploited in the
accumulation of THC, under open circuit conditions and using a 5 min pre-concentration time.
By testing the sensor in water and synthetic saliva samples of known THC concentrations,
analytical practical lower limits of detection (LODs) of 0.50 M and 0.10 M were obtained for
THC in stationary and stirred aqueous borate buffer solutions, respectively, and 0.50 M for
THC in stationary synthetic saliva solutions. Theoretical LOD values of 0.48 nM and 0.41 nM
were also calculated for the stationary and stirred buffer systems respectively. As Table 1 clearly
demonstrates, the theoretical limits of detection here determined are much lower than those
reported in the literature. Importantly, the practical limit of detection of 0.50 M determined in
synthetic saliva, and which corresponds to an observed signal, is comparable to theoretical
literature values, usually calculated. The limit of detection of 0.50 M is also practically useful
in terms of road side detection, this also clearly illustrating the value of absorptive stripping
voltammetry. This work opens up further studies into different ways of taking advantage of the
properties of carbon paste electrodes [31].
Competing interests
Both authors contributed equally to this work and have read and approved of the final
manuscript.
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Synthesis and biological evaluation of new nanosized aromatic polyamides containing
amido- and sulfonamidopyrimidines pendant structures
Hammed H A M Hassan1*, Elsayed M E Mansour1, Asmaa M S Abou Zeid1, Ehab R El-
Helow2, Amel F Elhusseiny1 and Raafat Soliman3
*Corresponding author: Hammed H A M Hassanhamed.hassan@alexu.edu.eg
Author Affiliations
1
Department of Chemistry, Faculty of Science, Alexandria University, Ibrahimia, Alexandria
21321, Egypt
2
Department of Microbiology and Immunology, Faculty of Pharmacy, Pharos University,
Canal El Mahmoudia Street, Alexandria 21311, Egypt
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria, Egypt
Received: 12 April 2015
Accepted: 5 August 2015
2015 Hassan et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0
changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Formula display:
Abstract
Background
Antibiotics are biocides or products that inhibit the growth of microorganisms in the living cells
and there are extensive works directed to develop efficient antimicrobial agents. The
sulfonamide-containing polymers have great potential to resist gram-positive or gram-negative
bacterial and fungal attacks. As a therapeutic agent, the sulfonamides have been reported as
antitumor and antimicrobial agents against bacteria, being more potent against gram positive
rather than gram negative strains. Design of new classes of inhibitors bearing fluorescent tails, as
therapeutic and imaging agents, is currently an active area of research. Here, we describe the
synthesis of a new family of polyamides based on chlorophenyl-3,5-diaminobenzamides, methyl
substituted pyrimidinoamido-3,5-diaminobenzamides and methyl substituted
pyrimidinosulfonamido-3,5-diaminobenzamides and evaluation of their thermal, optical and
antimicrobial properties.
Results
We report the synthesis of a new series of nanosized polyamides containing bioactive pendent
structures. The spherical nanosized polymer particles are soluble in many organic solvents and
exhibited emissions ranging from blue to orange wavelength depending on the nature of the
signaling unit. Pyrimidine- and p-chloroaromatic containing polymers exhibited higher
bioactivity than that contain the sulfonamide group. The amidopyrimidine polymers exhibited
remarkable antifungal and antibacterial activity and thus, these types of polymers are promising
candidates for biomedical applications.
Conclusions
The SEM analysis indicated that most of the polyamides were organized as well defined nano
sized spheres, but in certain derivatives small amount of aggregated nanospheres were also
observed. Thermal analyses were studied up to 700 C and results showed comparable thermal
behavior. The optical results revealed that polymeric series (A) exhibited orange emission, series
(B) showed green emission while series (C) exhibited yellow and blue emissions.
Benzene/pyridine structure interchange resulted in red shifted peaks attributed to the localized
lone pair of electrons on a nitrogen atom which offer a greater electron affinity and better
electron-transporting properties. The amido- and sulfonamide pyrimidine containing polymers
exhibited the most potent antimicrobial activity. Relative to the reference Gentamicin, the
polymer 54 exhibited comparable antibacterial activity against gram negative bacteria.
Analogues 52 and 57 exhibited remarkable antibacterial activities compared to the references
used. Thus, these polyamides are likely to be promising broad spectrum antibacterial agents and
deserve further investigation at the molecular level.
Keywords:
Synthesis; Polymers; Sulfonamides; Pyrimidines; Microbial activity
Graphical abstract
Background
Polymerdrug structures are currently known constructs that chemically combine the bioactive
part with a specific region of the polymer to ensure its delivery to the targeted intracellular
compartment [1]. Several synthetic approaches have been published to bond the polymer and
drug either in the polymer backbone or in the side-chain [2][4]. The development of longer term
bioactive antimicrobial polymers is a research area focused on solving microorganisms
contamination problems [5]. Polymer modifications to achieve such activity include
incorporation of known antimicrobial compounds such as hydantoins, glycolylureas,
imidazolidinones and oxazolidinones [6][8]. In addition, several studies have reported the
incorporation of a pyrimidine ring into the polymeric backbone. Incorporation of pyrimidine
nuclei modify the polymers solubility and processability due to the possibility of protonation
and/or alkylation of the lone pair. Moreover, the electronegative nitrogen atoms offers many
substituted pyrimidine structures through direct substitution reactions [9][12].
Sulfonamides have been used in therapeutics for many years [13], [14]. The sulfonamide
derivatives have been reported to show substantial antitumor activity in vitro and/or in vivo[15]
[18], HIV protease inhibitors [19], [20] and cell entry [21]. The polysulfonamide is an active
agent that shields the toxic polycations. The copolymers possess higher activity toward fungi
than against bacteria and being more gram positive rather than gram negative as it is common. A
novel strategy for cancer treatment based on a new class of inhibitors bearing fluorescent tails is
currently an active research area for use therapeutic and imaging agents for poorly responsive
tumors to classical chemo- and radiotherapies. For instance, a bioactive novel fluorescent fluoro
poly(amidesulfonamide)s possessed distinctive structure as well as unique properties were
reported [22].
We previously succeeded in preparing nanosized aromatic polyamides with remarkable electrical
and biomedical properties [23][29]. Herein, we describe the preparation of novel nanosized
aromatic polyamides with bioactive pendant structures comprised of substituted pyrimidines that
act as signaling units due to their fluorescent and chromogenic characteristics. We report the
synthesis of novel diamine types derived from isomeric chlorophenyl-3,5-dinitrobenzamides,
isomeric methyl substituted pyrimidinoamido-3,5-dinitrobenzamides and isomeric methyl
substituted pyrimidinosulfonamido-3,5-dinitrobenzamides. Subsequent reactions of these
diamines with the readily available isophthaloyl chloride or pyridine-2,6-dicarbonyl dichloride
furnished a new series of bioactive, fluorescent aromatic polyamides containing chloroaromatic,
pyrimidinoamido-, pyrimidinesulfonamido pendent structures, respectively. Evaluation of
thermal, optical and antimicrobial properties of the prepared polymers are also described.
Experimental
General
Melting points were determined with an electrothermal melting point apparatus and are not
corrected. Infrared spectra (IR, KBr pellets; 3 mm thickness) were recorded on a Perkin-Elmer
Infrared Spectrophotometer (FT-IR 1650). All spectra were recorded within the wave number
range of 6004,000 cm 1 at 25 C. 1 H-NMR and 13 C-NMR spectra were recorded using the
JEOL 500 mHZ spectrometer operating in DMSO-d 6 and expressed on the scale ppm.
Absorption spectra were measured with a UV 500 UVvis spectrometer at room temperature (r.t)
in DMSO with a polymer concentration of 2 mg/10 ml. Inherent viscosities (inh ) were measured
at a concentration of 0.5 g/100 dL in DMSO at 30 C by using an Ubbelohde Viscometer.
Differential thermo gravimetric (DTG) analyses were carried out in the temperature range from
20 to 700 C in a steam of nitrogen atmosphere by a Shimadzu DTG 60H thermal analyzer. The
experimental conditions were: platinum crucible, nitrogen atmosphere with a 30 ml/min flow
rate and a heating rate 20 C/min. Thermo gravimetric (TGA) analyses were carried out using
SDTQ600-V20.5-Build-15. (DTG), (TGA) and elemental analyses were performed at the
Microanalytical Unit, Cairo University. The morphologies of polymer nanoparticles were
observed by Scanning Electron Microscope (SEM) (JEOL-JSM5300), at the E-Microscope Unit;
Faculty of Science, Alexandria University. The samples were sonicated in de-ionized water for
5 min and deposited onto carbon coated copper mesh and allowed to air-dry before the
examination. The antimicrobial activities were carried out using diffusion agar techniques and
the evaluation of cytotoxicity against HepG-2, HCT-116 and MCF-7 cell lines were performed at
the antimicrobial unit and the regional center of mycology and biotechnology, Al-Azhar
University, Cairo.
Synthesis of 3,5-diaminobenzamide containing pendent aromatic structures 812, 16
18 and 2224 (General method)
The appropriate commercial amine 37, 1820 and 2729 (13.05 mmol) dissolved in DMF
(20 ml) was treated with 3,5 dinitrobenzoyl chloride 2 (13.05 mmol). The mixture was stirred for
20 h at r.t and then it was poured into cold water and filtered and the obtained 3,5-
dinitrobenzamides were dried in a vacuum oven at 60 C. The following data were recorded: 3,5-
Dinitro-N-phenylbenzamide8: Yield: (3 g, 80 %), m.p. 226 C. IR (, cm 1 ): 3,461, 3,290
(NH str ), 3,104 (CH str arom), 1,653 (C=O str amide), 1,600 (C=C str arom), 1,537 (NO 2asym str ),
1,494, 1,441, 1,340 (NO 2sym str ), 1,272, 1,162, 1,108, 1,076, 948, 916, 862, 816, 760, 726, 692,
568, 512. 1 H-NMR (500 MHz, DMSO): 9.14 (s, 1H, CONH), 9.11 (s, 2H, H5, H6 arom), 8.97
(s, 1H, H2 arom.), 7.767.13 (m, 5H, C6H5). Elemental analysis calculated for C 13 H 9 N 3 O 5 :
C, 54.35; H, 3.13; N, 14.63. Found: C, 54.71; H, 3.50; N, 14.91.
N-(2-Chlorophenyl)-3,5-dinitrobenzamide9: Yield: (6.1 g, 73 %), m.p. 204 C. IR (, cm 1 ):
3,448, 3,253 (NH str ), 3,099 (CH str arom), 1,657 (C=O str amide), 1,589 (C=C str arom), 1,537
(NO 2asym str ), 1,471, 1,436, 1,343 (NO 2sym str ), 1,307, 1,270, 1,163, 1,106, 1,077, 917, 823, 765
(CCl str ), 748, 676, 572. Elemental analysis calculated for C 13 H 8 ClN 3 O 5 : C, 48.50; H,
2.48; N, 13.06. Found: C, 48.21; H, 2.63; N, 13.29.
N-(3-Chlorophenyl)-3,5-dinitrobenzamide10: Yield: (6.8 g, 81.2 %), m.p. 219 C. IR (, cm 1 ):
3,429, 3,281 (NH str ), 3,100 (CH str aromatic), 1,663 (C=O str amide), 1,628, 1,595
(C=C strarom), 1,541 (NO 2asym str ), 1,477, 1,421, 1,344 (NO 2sym str ), 1,304, 1,257, 1,163, 1,079,
1,005, 917, 883 (CCl str ), 833, 790, 726, 689, 567, 534. Elemental analysis calculated for
C 13 H 8 ClN 3O 5 : C, 48.50; H, 2.48; N, 13.06. Found: C, 48.73; H, 2.66; N, 13.32.
N-(4-Chlorophenyl)-3,5-dinitrobenzamide11: Yield: (2.6 g, 62 %), m.p. 198 C. IR (, cm 1 ):
3,419, 3,270 (NH str ), 3,182, 3,097 (CH str arom), 2,920, 1,653 (C=O str amide), 1,598
(C=C strarom), 1,543 (NO 2asym str ), 1,493, 1,397, 1,343 (NO 2symstr ), 1,263, 1,163, 1,164, 1,090,
1,013, 955, 918, 869 (CCl str ), 827, 707, 509. Elemental analysis calculated for
C 13 H 8 ClN 3 O 5 : C, 48.50; H, 2.48; N, 13.06. Found: C, 48.22; H, 2.18; N, 13.36.
N-(4-(N-(2-Chlorophenyl)sulfamoyl) phenyl)-3,5-dinitrobenzamide12: Yield: (3.6 g, 57 %), m.p.
210 C. IR (, cm 1 ): 3,361 (NH str SO 2 NH), 3,254 (NH str CONH), 3,090 (CH str arom),
1,691, 1,628 (C=O str amide), 1,593 (C=C str arom), 1,537 (NO 2 str ), 1,482, 1,398, 1,339 (SO 2
asymstr), 1,264, 1,158 (SO 2symstr ), 1,087, 912, 835, 759 (C-Cl str ), 724, 684, 638, 565, 449.
Elemental analysis calculated for C 19 H 13 ClN 4 O 7 S: C, 47.84; H, 2.73; N, 11.75; S, 6.71.
Found: C, 47.37; H, 2.99; N, 11.47; S, 6.51.
3,5-Dinitro-N-(pyrimidin-2-yl) benzamide21: Yield: (3.3 g, 53 %), m.p. 175 C. IR (, cm 1 ):
3,307 (NH str ), 3,091 (NH str ), 2,961 (CH str arom), 2,882, 2,677, 2,540, 1,854, 1,705, 1,627
(C=O str amide), 1,543 (NO 2asymstr ), 1,470, 1,415, 1,350 (NO 2symstr ), 1,284 (CN str arom),
1,179, 1,078, 922, 799, 725, 695, 642, 532. Elemental analysis calculated for C 11 H 7 N 5 O 5 : C,
45.67; H, 2.42; N, 24.20. Found: C, 45.36; H, 2.73; N, 24.51.
N-(4-Methylpyrimidin-2-yl)-3,5-dinitrobenzamide22: Yield: (3.4 g, 52 %), m.p. 200 C. IR (,
cm1 ): 3,332 (NH str ), 3,091 (NH str ), 3,006 (CH str arom), 2,962, 2,881 (CH str ), 2,676, 2,539,
1,967, 1,852, 1,705, 1,628 (C=O str amide), 1,600 (C=C str arom), 1,544 (NO 2asymstr ), 1,470,
1,414, 1,350 (NO 2symstr ), 1,284 (C-N str arom), 1,179, 1,076, 921, 807, 783, 725, 694, 643,
531.1 H NMR (500 MHz, DMSO) ; 9.16 (1H, s, CONH), 9.11 (2H, s, H5 and H6 arom), 9.04
(1H, s, H2 arom), 8.17 (1H, d, pyrimidine H6 ), 6.88 (1H, d, pyrimidine H5), 2.43 (3H, s,
CH 3 ). Elemental analysis calculated for C 12 H 9 N 5 O 5 : C, 47.50; H, 2.97; N, 23.10. Found: C,
47.81; H, 2.63; N, 22.84.
N-(4,6-Dimethylpyrimidin-2-yl)-3,5-dinitrobenzamide23: Yield: (3.5 g, 64 %), m.p. 168 C. IR
(, cm 1 ): 3,343 (NH str ), 3,093(NH str ), 3,028 (CH str arom), 2,881 (CH str ), 2,677, 2,540,
1,974, 1,701, 1,626 (C=O str amide), 1,541(NO 2asym str ), 1,469, 1,417, 1,348 (NO 2symstr ), 1,285,
1,181, 1,077, 925, 834, 787, 724, 696, 634, 524. Elemental analysis calculated for
C 13 H 11 N 5 O5 : C, 49.21; H, 3.47; N, 22.08. Found: C, 49.48; H, 3.71; N, 21.92.
3,5-Dinitro-N-(4-(N-pyrimidin-2-yl sulfamoyl) phenyl) benzamide30: Yield: (2.6 g, 68 %), m.p.
291 C. IR (, cm 1 ): 3,399 (NH str SO 2 NH), 3,102 (NH str CONH), 3,040 (CH str arom), 2,936,
2,868, 2,811, 2,732, 1,682 (C=O str amide), 1,626, 1,587 (C=C str arom), 1,537 (NO 2 str ), 1,492,
1,444, 1,406, 1,342 (SO 2 asymstr ), 1,266 (CN str arom), 1,165 (SO 2 symstr ), 1,091, 1,001, 948, 921,
838, 798, 724, 680, 644, 570, 518. 1 H-NMR (500 MHz, DMSO) 9.136 (s, 1H, CONH), 9.11
(s, 2H, H5, H6 arom), 8.8 (s, 1H, H2 arom), 8.47 (m, 2H, H4 and H6 arom), 7.98 (m, 2H, H2
arom), 7.84 (m, 2H, H3 arom), 7.02 (m, 1H, H5 arom), 4.01 (s, 1H, SO 2 NH). MS-EI (m/z):
444 (4 M+ ), 410 (19), 407 (79), 361 (7), 304 (8), 277 (46), 249 (100), 232 (27), 205 (95), 152
(46), 140 (17), 127 (20), 77 (7), 29 (92). Elemental analysis calculated for C 17 H 12 N 6 O 7 S.
DMF: C, 48.54; H, 4.89; N, 18.11; S, 5.18. Found: C, 48.06; H, 3.62; N, 18.07; S, 6.47.
N-(4-(N-(4-Methylpyrimidin-2-yl) sulfamoyl) phenyl)-3,5-dinitrobenzamide31: Yield: (2.9 g,
73 %), m.p. 258 C. IR (, cm 1 ): 3,453 (NH str SO 2 NH), 3,401 (NH str CONH), 3,100 (C
H str arom), 2,859 (CH str ), 2,776, 1,685 (C=O str amide), 1,597, 1,541 (NO 2str ), 1,499, 1,446,
1,403, 1,345 (SO 2 asym str ), 1,289, 1,269 (CN str arom), 1,242, 1,209, 1,159 (SO 2symstr ), 1,086,
966, 917, 890, 840, 795, 727, 677, 577. Elemental analysis calculated for C 18 H 14 N 6 O 7 S: C,
47.16; H, 3.06; N, 18.34; S, 6.98. Found: C, 47.43; H, 3.39; N, 18.67; S, 7.23.
N-(4-(N-(4,6-Dimethylpyrimidin-2-yl) sulfamoyl) phenyl)-3,5-dinitrobenzamide32: Yield: (3.5 g,
57 %), m.p. 238 C. IR (, cm 1 ): 3,438 (NH str SO 2 NH), 3,111 (NH str CONH), 2,921 (C
H strarom), 2,854 (CH str ), 1,682 (C=O str amide), 1,628, 1,599 (C=C str arom), 1,541 (NO 2str ),
1,436, 1,401, 1,345 (SO 2 asymstr ), 1,265 (CN str arom), 1,159 (SO 2 sym str ), 1,141, 1,082, 1,031,
975, 919, 866, 842, 782, 715, 668, 587. Elemental analysis calculated for C 19 H 16 N 6 O 7 S: C,
48.30; H, 3.39; N, 17.79; S, 6.78. Found: C, 47.98; H, 2.89; N, 17.43; S, 7.02.
The above described 3,5-Dinitrobenzamide derivatives (3 g) dissolved in 30 ml of ethanol were
treated with 100 mg of Pd/C (10 %). Hydrazine hydrate (15 ml) was added dropwise over a
period of 1 h and the mixture was heated at 90 C for 15 h. The catalyst was removed by
filtration and the filtrate was concentrated under vacuum to dryness. The obtained solid was
dried in a vacuum oven at 60 C.
3,5-Diamino-N-phenylbenzamide13: Yield: 80 %, m.p. 197 C. IR (, cm 1 ): 3,437, 3,384

(NH 2 str ), 3,282 (NH str ), 3,057 (CH str arom), 3,015, 2,923, 1,652 (C=O str amide), 1,595
(C = C strarom), 1,532 (NH 2 ), 1,498, 1,464, 1,435, 1,355 (CN str ), 1,314, 1,255, 1,185, 1,029,
1,001, 855, 770, 749, 711, 683, 604, 564, 526, 476. 1 H-NMR (500 MHz, DMSO) 9.14 (s, 1H,
CONH), 6.997.702 (m, 5H, ArH), 6.45 (s, 2H, H5, H6 arom), 6.33 (s, 2H, NH 2 ), 6.03 (s, 1H,
H2 arom). Elemental analysis calculated for 8; C 13 H 13 N 3 O: C, 68.72; H, 5.72; N, 18.50.
Found: C, 68.44; H, 5.37; N, 18.28.
N(2-Chlorophenyl)-3,5-diaminobenzamide14: Yield: 85 %, m.p. 155 C. IR (, cm 1 ): 3,454,
3,415, 3,369 (NH 2str ), 3,250 (NH str ), 3,081 (CH str arom), 2,924, 1,680 (C=O str amide), 1,641,
1,587 (C=C str arom), 1,522 (NH 2 ), 1,436, 1,348 (CN str ), 1,231, 1,053, 996, 936, 873, 807, 751,
733, 659, 551, 518, 436. Elemental analysis calculated for 14; C 13 H 12 ClN 3 O: C, 59.65; H,
4.59; N, 16.06. Found: C, 59.82; H, 4.76; N, 15.78.
3,406, 3,362 (NH 2str ), 3,225 (NH str ), 3,089 (CH str ), 2,924, 1,674 (C=O str , amide), 1,634,
1,593 (CH str arom), 1,520 (NH 2 ), 1,477, 1,419, 1,347 (CN str ), 1,274, 1,242, 1,107, 1,079, 996,
932, 905, 871, 815, 774, 730, 682, 598, 536, 437. Elemental analysis calculated for 15;
C 13 H 12ClN 3 O: C, 59.65; H, 4.59; N, 16.06. Found: C, 59.42; H, 4.92; N, 16.39.
3,394 (NH 2str ), 3,280 (NH str ), 1,640 (C=O str amide), 1,594 (CH str arom), 1,526 (NH 2 ), 1,491,
1,395, 1,353 (CN str ), 1,309, 1,246, 1,190, 1,092, 1,010, 855, 710, 494, 428. 1 H NMR
(500 MHz, DMSO) ; 9.15 (1H, s, CONH), 8.01 (2H, d, H2 ArCl), 7.69 (2H, d, H3 ArCl),
6.29 (2H, s, H5 and H6 arom), 6.29 (4H, s, NH 2 ), 5.49 (1H, s, H2 arom). Elemental analysis
calculated for 16; C 13 H12 ClN 3 O: C, 59.65; H, 4.59; N, 16.06. Found: C, 59.32; H, 4.86; N,
15.81.
N(4-(N-(2-Chlorophenyl)sulfamoyl)phenyl)-3,5-diaminobenzamide17: Yield: 41 %, m.p. 154 C.
IR (, cm 1 ): 3,425, 3,360 (NH 2 str ), 1,670 (C=O str amide), 1,592 (C = C str arom), 1,518
(NH 2 ), 1,483, 1,400, 1,324 (SO 2 asym str ), 1,229, 1,189, 1,156 (SO 2 symstr ), 1,094, 1,057, 999,
921, 868, 831, 760 (CCl str ), 727, 682, 595, 565. Elemental analysis calculated for 17;
C 19 H 17 ClN 4O 3 S: C, 54.74; H, 4.08; N, 13.44; S, 7.68. Found: C, 54.33; H, 4.41; N, 13.73; S,
7.39.
3,5-Diamino-N-(pyrimidin-2-yl) benzamide24: Yield: 92 %, m.p. 200 C. IR (, cm 1 ): 3,697,
3,453, 3,369 (NH 2str ), 3,086 (NH str ), 2,924 (CH str arom), 2,656, 1,631 (C=O str amide), 1,572
(C=C str arom), 1,526 (NH 2 ), 1,463, 1,391, 1,340 (CN str ), 1,295, 1,081, 999, 948, 917, 865,
797, 736, 657, 606, 544. Elemental analysis calculated for 24; C 11 H 11 N 5 O: C, 57.64; H, 4.80;
N, 30.57. Found: C, 57.34; H, 4.78; N, 30.86.
N(4-Methylpyrimidin-2-yl)-3,5-diaminobenzamide25: Yield: 63 %, m.p. 118 C. IR (, cm 1 ):
3,726, 3,340 (NH 2str ), 3,224 (NH str ), 2,966 (CH str arom), 2,629, 2,076, 1,626 (C=O str amide),
1,567 (NH 2 ), 1,469, 1,400 (CN str ), 1,157, 998, 941, 859, 762, 674, 562, 498. 1 HNMR
(500 MHz, DMSO) ; 9.15 (1H, s, CONH), 8.23 (1H, d, pyrimidine H6), 6.88 (1H, d,
pyrimidine H5), 6.38 (2H, s, H5 and H6 arom), 6.25 (4H, s, NH 2 ), 5.97 (1H, s, H2 arom), 2.391
(3H, s, CH 3 ). Elemental analysis calculated for 25; C 12 H 13 N 5 O: C, 59.26; H, 5.35; N, 28.80.
Found: C, 59.63; H, 5.71; N, 29.03.
N(4,6-Dimethylpyrimidin-2-yl)-3,5-diaminobenzamide26: Yield: 84 %, m.p. 186 C. IR (,
cm 1 ): 3,697, 3,459, 3,379 (NH 2 str ), 3,087 (NH str ), 1,625 (C=O str amide), 1,575
(C=C str arom), 1,525 (NH 2 ), 1,389, 1,337 (CN str ), 1,081, 948, 882, 792, 735, 656, 606, 438.
Elemental analysis calculated for 26; C 13 H 15 N 5 O: C, 65.70; H, 5.83; N, 27.23. Found: C,
65.47; H, 5.52; N, 27.46.
3,5-Diamino-N-(4-(N-pyrimidin-2-yl)sulfamoyl)phenyl)benzamide33: Yield: 46 %, m.p. 242 C.
IR (, cm 1 ): 3,448 (NH str SO 2 NH), 3,422, 3,341 (NH 2str ), 3,204 (NH str CONH),
1,635(C=O stramide), 1,609 (CH str arom), 1,523 (NH 2 ), 1,395, 1,356 (SO 2 asymstr ), 1,311, 1,253
(CN strarom), 1,165, 1,135 (SO 2symstr ), 1,087, 1,012, 822, 779, 756, 692, 633, 553. 1 H-NMR
(500 MHz, DMSO): 9.14 (s, 1H, CONH), 8.47 (m, 2H, H4, H6 arom), 7.96 (m, 2H H2,
ArH), 7.84 (m, 2H, H3, ArH), 7.01 (m, 1H, H5 arom), 6.48 (s, 2H, H5, H6 arom), 6.31 (s, 2H,
NH 2 ), 5.98 (s, 1H, H2 dinitro arom), 4.01 (s, 1H, SO 2 NH). Elemental analysis calculated
for 33; C 17 H 16 N 6 O 3 S: C, 53.12; H, 4.167; N, 21.87; S, 8.33. Found: C, 53.28; H, 4.36; N,
21.51; S, 8.12.
N(4-(N-(4-Methylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-diaminobenzamide34: Yield: 80 %, m.p.
216 C. IR (, cm 1 ): 3,731, 3,464 (NH str SO 2 NH), 3,412, 3,343 (NH 2 str ), 3,276, 3,082
(NH strCONH), 1,928, 1,815, 1,667 (C=O str amide), 1,627, 1,590 (CH str arom), 1,527 (NH 2 ),
1,398, 1,332 (SO 2 asymstr ), 1,251 (CN str arom), 1,192, 1,154 (SO 2 symstr ), 1,094, 873, 835, 688,
596, 545. Elemental analysis calculated for 34; C 18 H 18 N 6 O 3 S: C, 54.27; H, 4.52; N, 21.10;
S, 8.04. Found: C, 54.52; H, 4.86; N, 21.39; S, 8.40.
N(4-(N-(4,6-Dimethylpyrimidin-2-yl) sulfamoyl) phenyl)-3,5-diaminobenzamide35: Yield: 40 %,
m.p. 172 C. IR (, cm 1 ): 3,561, 3,457 (NH str SO 2 NH), 3,397, 3,361 (NH 2str ), 3,288
(NH strCONH), 1,666 (C=O str amide), 1,599 (C=C str arom), 1,531 (NH 2 ), 1,401, 1,368 (SO 2
asymstr ), 1,313 (CN str ), 1,258 (CN str arom), 1,186, 1,139 (SO 2symstr ), 1,079, 1,029, 962, 848,
780, 713, 677, 588, 548. Elemental analysis calculated for 35; C 19 H 20 N 6 O 3 S: C, 55.34; H,
4.85; N, 20.39; S, 7.76. Found: C, 55.58; H, 5.09; N, 20.61; S, 7.99.
Reaction of isophthaloyl chloride with 3,5-diaminobenzamide containing chloro aromatic
pendant structures 1317: synthesis of polyamides 3640 (General method)
The readily available isophthaloyl chloride (2.20 mmol) was slowly added to a stirred solution of
the appropriate diamine 1317 (2.20 mmol) dissolved in 10 ml DMA at 0 C (ice bath). The
mixture was stirred overnight at r.t then it was poured into iced water. The precipitate was
collected by filtration, washed thoroughly with water, ethanol, and water again and dried in a
vacuum oven at 80 C.
Preparation of polymer 36
Following the general method described above, isophthaloyl dichloride reacted with 3,5-
diamino-N-phenylbenzamide 13 to give the polyamide 36. The following data were recorded:
Yield: 0.8 g, 97 %, m.p >300 C, inh = 1.48 dL/g. IR (, cm 1 ): 3,727, 3,275 (NH str ), 3,094
(CH str arom), 2,924, 1,663 (C=O str amide), 1,600 (C=C str arom), 1,535, 1,441, 1.327, 1,239,
1,077, 1,010, 871, 820, 755, 715, 686, 585, 530. Elemental analysis calculated for the
polyamide 36 (C 21 H 15 N3 O 3 ) n .H 2 O: C, 67.20; H, 4.53; N, 11.20. Found: C, 67.51; H, 4.78;
N, 11.43.
Following the general method described above, isophthaloyl dichloride reacted N-(2-chloro
phenyl)-3,5-diaminobenzamide 14 to give the polyamide 37. The following data were recorded:
Yield: 0.39 g, 50 %, m.p >300 C, inh = 0.14 dL/g. IR (, cm 1 ): 3,387, 3,235 (NH str amide),
3,081 (CH str arom), 1,688, 1,660 (C=O str amide), 1,590 (C=C str arom), 1,532, 1,437, 1,343,
1,311, 1,237, 1,133, 1,085, 1,057, 947, 896, 817, 733 (CCl str ), 688, 593, 551, 441. Elemental
analysis calculated for polyamide 37 (C 21 H 14 ClN 3 O 3 ) n .H 2 O: C, 61.50; H, 3.90; N, 10.26.
Found: C, 61.22; H, 4.31; N, 10.59.
Following the general method described above, isophthaloyl dichloride reacted N-(3-
chlorophenyl)-3,5-diaminobenzamide 15 to give the polyamide 38; yield: 0.48 g, 62 %, m.p
>300 C, inh = 0.28 dL/g. IR (, cm 1 ): 3,291 (NH str amide), 3,187, 3,081 (CH str arom),
3,009, 2,883, 2,665, 2,553, 1,689, 1,661 (C=O str amide), 1,593 (C=C str arom), 1,533, 1,480,
1,421, 1,342, 1,285, 1,166, 1,080, 998, 893, 825 (CCl str ), 780, 730, 685, 598, 539, 439.
Elemental analysis calculated for polyamide 38 (C 21 H 14 ClN 3 O 3 ) n .H 2 O: C, 61.50; H, 3.90;
N, 10.26. Found: C, 61.79; H, 3.62; N, 10.54.
Following the general method described above, isophthaloyl dichloride reacted N-(4-
chlorophenyl)-3,5-diaminobenzamide 16 to give the polyamide 39; yield: 0.75 g, 95 %, m.p
>300 C, inh = 0.47 dL/g. IR (, cm 1 ): 3,298 (NH str amide), 3,109 (CH str aromatic), 1,661
(C=O str amide), 1,599 (C=C str arom), 1,535, 1,492, 1,445, 1,398, 1,330, 1,242, 1,090, 1,009,
874 (CCl str ), 825, 719, 591, 504, 432. Elemental analysis calculated for
polyamide 39 (C 21 H 14ClN 3 O 3 ) n . H 2 O: C, 61.50; H, 3.90; N, 10.26. Found: C, 61.83; H,
4.28; N, 10.61.
Following the general method described above, isophthaloyl dichloride reacted N-(4-(N-(2-
chlorophenyl)sulfamoyl) phenyl)-3,5-diaminobenzamide 17 to give the polyamide 40; yield:
0.65 g, 96 %, m.p >300 C, inh = 0.89 dL/g. IR (, cm 1 ): 3,339 (NH str amide), 3,105 (C
H strarom), 2,925, 1,924, 1,665 (C=O str amide), 1,594 (C=C str arom), 1,528, 1,484, 1,446, 1,401,
1,330 (SO 2 asym str ), 1,250, 1,158 (SO 2 sym str ), 723, 686, 563. Elemental analysis calculated for
polyamide 40 (C 27 H 19 ClN 4 O 5 S) n .H 2 O: C, 57.40; H, 3.72; N, 9.92; S, 5.67. Found: C,
57.79; H, 3.38; N, 9.56; S, 5.65.
Reaction of pyridine 2,6-dicarbonyl dichloride with 3,5-diaminobenzamide containing
chloroaromatic pendant structures1317: synthesis of polyamides 4145
The readily available pyridine 2,6-dicarbonyl dichloride (2.20 mmol) was the appropriate
diamine13-17 (2.20 mmol) following the above mentioned general method.
Following the general method described above, pyridine 2,6-dicarbonyl dichloride reacted with
3,5-diamino-N-phenylbenzamide 13 to give the polyamide 41. The following data were
recorded: Yield: 0.8 g, 96 %, m.p >300 C, inh = 1.44 dL/g. IR (, cm 1 ): 3,427
(NH str amide), 2,968 (CH str arom), 2,924, 1,675 (C=O str amide), 1,600 (C=C str arom), 1,531,
1,444, 1,328 (CN strarom), 1,238, 1,184, 1,135, 1,076, 1,047, 997, 872, 749, 680, 530. Elemental
analysis calculated for the polyamide 41 (C 20 H 14 N 4 O 3 ) n .H 2 O: C, 63.80; H, 4.25; N, 14.89.
Found: C, 63.52; H, 4.61; N, 14.51.
Following the general method described above, pyridine 2,6-dicarbonyl dichloride reacted
with N-(2-chlorophenyl)-3,5-diaminobenzamide 14 to give the polyamide 42; yield: 0.38 g,
48 %, m.p >300 C, inh = 0.47 dL/g. IR (, cm 1 ): 3,785, 3,726, 3,698, 3,469, 3,406
(NH str aromatic), 3,312 (NH str amide), 2,926 (CH str arom), 1,688, 1,623 (C=O str amide), 1,591
(C=C str arom), 1,532, 1,438, 1,346 (CN str arom), 1,218, 1,135, 1,078, 1,034, 1,000, 942, 898,
845, 814,744 (C-Clstr ), 680, 555. Elemental analysis calculated for
polyamide 42 (C 20 H 13 ClN 4 O 3 ) n .H 2 O: C, 58.46; H, 3.65; N, 13.64. Found: C, 58.76; H,
3.34; N, 13.25.
47 %, m.p >300 C, inh = 0.52 dL/g. IR (, cm 1 ): 3,728, 3,427 (NH str arom), 3,298
(NH str amide), 3,107 (CH str arom), 1,677 (C=O str amide), 1,599 (C=C str arom), 1,533, 1,493,
1,449, 1,397, 1,310 (CN str arom), 1,239, 1,190, 1,142, 1,084, 1,004, 942, 873 (CCl str ), 828,
748, 678, 504. Elemental analysis calculated for the polyamide 43 (C 20 H 13 ClN 4 O 3 ) n .H 2 O:
C, 58.46; H, 3.65; N, 13.64. Found: C, 58.71; H, 3.89; N, 13.27.
85 %, m.p >300 C, inh = 0.72 dL/g. IR (, cm 1 ): 3,728, 3,298 (NH str amide), 3,106 (C
H str arom), 2,117, 1,988, 1,678 (C=O str amide), 1,599 (C=C str arom), 1,533, 1,493, 1,449,
1,397, 1,310 (CNstr arom), 1,238, 1,189, 1,142, 1,085, 1,004, 942, 872, 828 (CCl str ), 748, 678,
504. Elemental analysis calculated for the polyamide 44 (C 20 H 13 ClN 4 O 3 ) n .H 2 O: C, 58.46;
H, 3.65; N, 13.64. Found: C, 58.18; H, 3.26; N, 13.32.
with N-(4-(N-(2-chlorophenyl) sulfamoyl) phenyl)-3,5-diaminobenzamide 17 to give the
polyamide 45; yield: 0.66 g, 97 %, m.p >300 C, inh = 0.75 dL/g. IR (, cm 1 ): 3,266 (N
H str amide), 3,105 (CH str arom), 2,396, 1,781, 1,682 (C=O str amide), 1,595 (C=C str arom),
1,527, 1,485, 1,452, 1,400, 1,330 (SO 2asymstr ), 1,253, 1,222, 1,159 (SO 2symstr ), 1,091, 1,056,
1,000, 916, 837, 752, 725 (CCl str ), 683, 628, 562. Elemental analysis calculated for the
polyamide 45 (C 26 H 18 ClN 5O 5 S) n .H 2 O: C, 55.17; H, 3.50; N, 12.38; S, 5.66. Found: C,
55.49; H, 3.82; N, 12.59; S, 5.94.
Reaction of isophthaloyl chloride with 3,5-diaminobenzamide containing substituted
pyrimidine-2-yl pendant structures 2426: synthesis of polyamides 4648 (general method)
Isophthaloyl chloride (2.20 mmol) was treated with a solution of the appropriate diamine 24
26(2.20 mmol) following the general procedure described above.
Following the general method, isophthaloyl dichloride was treated with 3,5-diamino-N-
(pyrimidin-2-yl)benzamide 24 to furnish the polymer 46; yield: 0.66 g, 88 %, m.p
>300 C, inh = 0.80 dL/g. IR (, cm 1 ): 3,399 (NH str amide), 2,924 (CH str arom), 1,655
(C=O str amide), 1,536 (C=C strarom), 1,346, 1,267, 1,086, 904, 784, 733, 682, 594. Elemental
Found: C, 60.18; H, 3.61; N, 18.22.
Following the general method, isophthaloyl dichloride was treated with N-(4-methylpyrimidin -
2-yl)-3,5-diaminobenzamide 25 to furnish the polymer 47; yield: 0.7 g, 88 %, m.p
(C=O str amide), 1,610 (C=C str arom), 1,543, 1,448, 1,333, 1,231, 876, 772, 713, 597. Elemental
Found: C, 61.64; H, 4.58; N, 17.52.
Following the general method, isophthaloyl dichloride was treated with N-(4,6-dimethyl
pyrimidin-2-yl)-3,5-diaminobenzamide 26 to furnish the polymer 48; yield: 0.65 g, 83 %, m.p
(C=O str amide), 1,536 (C=C str arom), 1,435, 1,346, 1,268 (CN str arom), 1,087, 905, 785, 733,
682, 596. Elemental analysis calculated for the polyamide 48 (C 21 H 17 N 5 O 3 ) n .H 2 O: C,
62.20; H, 4.69; N, 17.28. Found: C, 62.60; H, 4.24; N, 17.57.
substituted pyrimidine-2-yl pendant structures 2426: synthesis of polyamides 49-51
Pyridine 2,6-dicarbonyl dichloride (2.20 mmol) was treated with a solution of the appropriate
diamine 2426 (2.20 mmol) in DMA as described earlier.
Following the described general method, pyridine 2,6-dicarbonyl dichloride was reacted with
3,5-diamino-N-(pyrimidin-2-yl) benzamide 24 to produce the polymer 49; yield: 0.74 g, 80 %,
m.p >300 C, inh = 0.32 dL/g. IR (, cm 1 ): 3,438 (NH str amide), 2,923 (CH str arom), 2,856,
1,696, 1,666 (C=O str amide), 1,629 (C=N str arom), 1,595 (C=C str arom), 1,536, 1,436, 1,346
(CN str arom), 1,278, 1,222, 1,136, 1,078, 1,000, 906, 843, 784, 742, 658, 601. Elemental
analysis calculated for polyamide 49 (C 18 H 12 N 6 O 3 ) n .H 2 O: C, 57.14; H, 3.70; N, 22.20.
Found: C, 57.48; H, 3.41; N, 22.52.
Following the described method, pyridine-2,6-dicarbonyl dichloride was treated with N-(4-
methylpyrimidin-2-yl)-3,5-diaminobenzamide 25 to give the polymer 50; yield: 0.8 g, 83 %, m.p
>300 C, inh = 0.54 dL/g. IR (, cm 1 ): 3,726, 3,435 (NH str amide), 2,924 (CH str arom),
2,858 (CH str ), 1,681 (C=O str amide), 1,607 (C=C str arom), 1,539, 1,453, 1,332 (CN str arom),
1,297, 1,221, 1,139, 1,078, 997, 873, 775, 743, 670, 617. Elemental analysis calculated for
polyamide 50 (C 19 H 14 N 6 O 3 ) n .H 2 O: C, 58.16; H, 4.08; N, 21.43. Found: C, 58.41; H, 4.39;
N, 21.16.
Following the method described earlier, pyridine-2,6-dicarbonyl dichloride was reacted with N-
(4,6-dimethylpyrimidin-2-yl)-3,5-diaminobenzamide 26 to give the polymer 51; yield: 0.75 g,
76 %, m.p >300 C, inh = 0.28 dL/g. IR (, cm 1 ): 3,444 (NH str arom), 3,294 (NH str amide),
3,093 (CH str arom), 2,926, 2,860 (CH str ), 1,695, 1,667 (C=O str amide), 1,595 (C=C str arom),
1,537, 1,434, 1,347 (CN str arom), 1,281, 1,223, 1,139, 1,079, 1,000, 906, 843, 786, 743, 660,
601. Elemental analysis calculated for the polyamide 51 (C 20 H 16 N 6 O 3 ) n .H 2 O: C, 59.10; H,
4.43; N, 20.69. Found: C, 59.38; H, 4.71; N, 20.37.
Reaction of isophthaloyl chloride with 3,5-diaminobenzamide containing substituted
sulfonamidopyrimidine-2-yl pendant structures3335: synthesis of polyamides 5254
Isophthaloyl chloride (2.20 mmol) was reacted with the appropriate diamine 3335 (2.20 mmol)
following the general method described above.
Following the general method described above, isophthaloyl dichloride was reacted with 3,5-
diamino-N-(4-(N-pyrimidin-2-ylsulfamoyl)phenyl)benzamide 33 to give the polymer 52; yield:
1.2 g, 91 %, m.p >300 C, inh = 1.16 dL/g. IR (, cm 1 ): 3,435 (NH str amide), 2,924
(CH strarom), 1,659 (C=O str amide), 1,597 (C=C str arom), 1,531, 1,443, 1,326 (SO 2asymstr ),
1,249, 1,154 (SO 2symstr ), 899, 830, 683, 593, 546. Elemental analysis calculated for the
polyamide 52(C 25 H 18 N 6 O 5 S) n .H 2 O: C, 56.39; H, 3.76; N, 15.79; S, 6.01. Found: C, 56.52;
H, 3.44; N, 15.63; S, 6.34.
Following the same described method, isophthaloyl dichloride was treated with N-(4-(N-(4-
methylpyrimidin-2-yl) sulfamoyl) phenyl)-3,5-diaminobenzamide 34 to furnish the polymer 53;
yield: 1 g, 80 %, m.p >300 C, inh = 0.70 dL/g. IR (, cm 1 ): 3,404 (NH str amide), 2,924
(CHstr arom), 1,665 (C=O str amide), 1,596 (C=C str arom), 1,531, 1,437, 1,400, 1,327
(SO 2asymstr ), 1,247, 1,154 (SO 2symstr ), 1,094, 898, 831, 684, 592, 546. Elemental analysis
calculated for the polyamide 53 (C 26 H 20 N 6 O 5 S) n .H 2 O: C, 57.14; H, 4.03; N, 15.38; S,
5.86. Found: C, 57.48; H, 4.36; N, 15.66; S, 6.09.
Following the same described method given above, isophthaloyl dichloride was reacted with N-
(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-diaminobenzamide 35 to produce the
polymer 54; yield: 0.6 g, 98 %, m.p >300 C, inh = 0.81 dL/g. IR (, cm 1 ): 3,432
(NH stramide), 2,924 (CH str arom), 1,678 (C=O str amide), 1,599 (C=C str arom), 1,534, 1,441,
1,301 (SO 2asymstr ), 1,259, 1,154 (SO 2symstr ), 1,081, 862, 723, 685, 587. Elemental analysis
calculated for the polyamide 43 (C 27 H 22 N 6 O 5 S) n .H 2 O: C, 57.86; H, 4.28; N, 15.00; S,
5.71. Found: C, 57.64; H, 4.59; N, 14.72; S, 5.43.
substituted sulfonamidopyrimidine-2-yl pendant structures 3335: synthesis of
polyamides 5557
Pyridine 2,6-dicarbonyl dichloride (2.20 mmol) was treated with s solution of the appropriate
diamine 2224 in DMA as described earlier.
Pyridine 2,6-dicarbonyl dichloride was treated with 3,5-diamino-N-(4-(N-pyrimidin-2-yl
sulfamoyl) phenyl)benzamide 33 following the described method mentioned earlier to give the
polymer 55; yield: 1.24 g, 95 %, m.p >300 C, inh = 0.24 dL/g. IR (, cm 1 ): 3,726, 3,435
(NH str amide), 2,923 (CH str arom), 2,856, 1,668 (C=O str amide), 1,595 (C=C str arom), 1,528,
1,447, 1,398, 1,321 (SO 2asymstr ), 1,247, 1,153 (SO 2symstr ), 1,091, 888, 834, 748, 681, 590.
Elemental analysis calculated for the polyamide 55 (C 24 H 17 N 7 O 5 S) n .H 2 O: C, 54.03; H,
3.56; N, 18.39; S, 6.00. Found: C, 54.32; H, 3.81; N, 18.65; S, 6.21.
Pyridine-2,6-dicarbonyl dichloride was reacted with N-(4-(N-(4-methylpyrimidin-2-
yl)sulfamoyl) phenyl)-3,5-diaminobenzamide 34 to give the polymer 56; yield: 1.2 g, 89 %, m.p
>300 C, inh = 0.35 dL/g. IR (, cm 1 ): 3,726, 3,437 (NH str amide), 2,924 (CH str arom), 2,857
(CH str ), 1,678 (C=O str amide), 1,595 (C=C str arom), 1,529, 1,449, 1,398, 1,322 (SO 2asymstr ),
1,248, 1,153 (SO 2symstr ), 1,089, 1,004, 895, 834, 739, 680, 593. Elemental analysis calculated
for polyamide 56 (C 25 H 19 N 7 O 5 S) n .H 2 O: C, 54.84; H, 3.84; N, 17.91; S, 5.85. Found: C,
55.09; H, 4.17; N, 17.62; S, 6.19.
Pyridine-2,6-dicarbonyl dichloride was treated with N-(4-(N-(4,6-dimethylpyrimidin-2-yl)
sulfamoyl) phenyl)-3,5-diaminobenzamide 35 to give the polymer 46; yield: 1.2 g, 87 %, m.p
(CH str ), 1,680 (C=O str amide), 1,597 (C=C str arom), 1,530, 1,444, 1,309 (SO 2asymstr ), 1,249,
1,151 (SO 2 asymstr ), 1,077, 1,003, 841, 787, 747, 679, 585. Elemental analysis calculated for
polyamide 57 (C26 H 21 N 7 O 5 S) n .H 2 O: C, 55.61; H, 4.10; N, 17.47; S, 5.70. Found: C, 55.90;
H, 4.36; N 17.73; S, 5.34.
Polymer particles synthesis (general Method)
The appropriate, readily available isophthaloyl dichloride, pyridine 2,6-dicarbonyl dichloride or
the prepared one 60 (0.5 mmol) and the appropriate diamine 1317, 2426, 3335 or 61
66(0.5 mmol) were separately dissolved in dioxane (50 mL). Distilled water (10 mL) was added
to the diamine and the entire solution was added to the acid chloride. The resulted turbid solution
was ultrasonicated at 42 kHz in a water bath for a period of 30 min. The polymer colloidal
solution was extracted by centrifugal separation for 15 min. at 6,000 rpm and the resulted
precipitate were carefully washed with methanol and water to purify the product of any
unreacted monomer. The polymer samples were then dried in a vacuum oven at 60 C for 10 h.
Results and discussions
Synthesis of 3,5-diaminobenzamide containing pendent aromatic structures 812, 16

18 and 2224
3,5-Diaminobenzamide containing pendent chloro aromatic structures 1812, Scheme 1, were
prepared by the reaction of 3,5-dinitrobenzoic acid 1 with thionyl chloride and the obtained acid
chloride 2 was then subsequently treated with a number of commercial substituted anilines 3
6 or 4-amino-N-(2-chlorophenyl) benzenesulfonamide 7, respectively, in DMF to furnish the
corresponding substituted 3,5-dinitrobenzamide 812. The FT-IR spectra exhibited bands in the
region 3,2533,361 cm 1 belong to the NH str absorption, a band at 1,650 cm 1 due to C=O
amide while the NO 2 bands appeared at 1,541 and 1,340 cm 1 , the SO 2 appeared at 1,339 and
1,158 cm 1 . The reaction of the dinitrobenzamides 812 with hydrazine hydrate/PdC (10 %)
furnished the corresponding 3,5-diaminobenzamides 1317, respectively, in good yields. The
FT-IR spectra of diamines 1317 showed absorption bands correspond to NH 2 in the region
3,4333,454 cm 1 and 3,3603,394 cm 1 , a characteristic band at 3,250 cm 1 due to the NH
while the C=O bands were in region 1,6401,680 cm 1 .
Scheme 1
Synthesis of 3,5-diaminobenzamides containing aromatic pendent structures 817, 2126 and30
35
Similarly, 3,5-diaminobenzamide containing pendent pyrimidine or sulfonamide pyrimidine 24
26and 3335 were prepared by reaction the acid chloride 2 with the amines 1820; namely: 2-
aminopyrimidine 18, 2-amino-4-methylpyrimidine 19, 2-amino-4,6-dimethylpyrimidine 20 or
aminosulfonamides 27-29 namely; sulfadiazine 27, sulfamerazine 28 and sulfamethazine 29,
respectively, in DMF. The IR spectra of the prepared dinitro compounds 2123 and 30
33exhibited bands at 3,100 cm 1 correspond to the amide NH; bands in the region 1,626
1,685 cm1 due to C=O amide, while the NO 2 bands appeared at 1,540 cm 1 . The obtained 3,5-
dinitrobenzamides were reduced using hydrazine hydrate/PdC (10 %) mixture following
standard procedure. The IR spectra of the diamines 2426 and 3335 showed absorption bands
correspond to the NH 2 at 3,453 and 3,361 cm 1 ; bands around 3,204 cm 1 due to the carbonyl
NH and the C=O amide bands were in region 1,6251,666 cm 1 . Physical properties of all new
compounds are recorded in the experimental section and the calculated analysis data are in good
agreement with the experimental one.
Synthesis of polyamides containing pendent chloro aromatic and pyrimidine- and
sulfonamidopyrimidine pendent structures 3645, 4651, 5257
The production of new aromatic polyamides containing chloro aromatic, pyrimidine- and
sulfonamidopyrimidine pendent structures, where the pendent groups act as signaling units due
to their fluorescent, chromogenic and biological characteristics and studying of their properties
are the objectives of our study. The important tasks in this study are to analyze and predict the
properties such as solubility, optical and fluorescence emission properties, biological activities
and thermal stability with respect to their chemical structures. The targeted polyamides 36
45, 4651, 5257 were synthesized in bulk scale by direct polycondensation of an equimolar
mixture of the readily available isophthaloyl dichloride or pyridine-2,6-dicarbonyl dichloride
with, respectively, the diamines 1317, 2426, 3335 in DMA solutions at 0 C (ice bath),
Fig. 1. The polyamides were obtained in moderate to good yields and their inherent viscosities
( inh ) are in the range of 0.241.48 dL/g. Physical properties of the new compounds are
recorded in the experimental section and the calculated analysis data are in good agreement with
the experimental one.
Fig. 1. Chemical structures of the polyamides 3645, 4651 and 5257
The synthesis of the nanosized aramides particles 3645, 4651 and 5257 was the next task.
Different solution techniques are known in the literature for the preparation of nanosized
particles, including emulsion, interfacial polycondensations or nanoprecipitation method [23]
[29]. The basic principle of the latter method is based on the interfacial deposition of a polymer
from solvent/non-solvent phases. Generally, the current series were prepared by ultrasonication
of 0.5 mmol of the appropriate diamine with 0.5 mmol of the acid chloride in a total of 115 ml
dioxane solution containing distilled water (15 ml) followed by centrifugal separation at
6,000 rpm for 30 min. The presence of water is necessary for controlling the particle shape and
as a reaction accelerator. As judged by SEM micrographs, Figs. 2 and 3, most polyamides were
obtained as well-separated spherical nanosized forms, nevertheless, there were some degree of
aggregation for those polymers containing pyridine and pyrimidine pendant groups. The
aggregate formation could be attributed to the molecular H-bond self-assembly via H-bond
directed organization of molecular precursors. The average diameters (standard deviation) of
some polymers were 39; 66.76 nm (28.36), 40; 198.86 (27.45), 41 92.31 nm (27.59) and 45;
209.27 nm (10.63); 46; 406.12 nm (39.12), 47; 205.6 nm (34.31), 48; 77.27 nm (25.6), 49;
48.29 nm (9.8), 50; 58.99 (13.37), 51; 61.08 nm (5.44); 54; 69.6 nm (13.43) and 57; 71.02 nm
(18.85), respectively.
Fig. 2. SEM images of the nanosized aramides 3941, 4546 and 49
Fig. 3. SEM images of the nanosized aramides 4748, 5051, 54 and57

Physical properties of the polymers
Solubility
The polyamides are readily soluble in polar aprotic solvents such as NMP, DMAc, DMF and
DMSO while insoluble in boiling alcoholic or halogenated solvents. The observed solubility of
the pyridine-containing polyamide compared to that polyamides containing phenylene moiety
could be attributed to the dipoledipole interaction of polymersolvent system. The pyrimidine-
containing polymers showed inferior (lower) solubility may be due to the presence of pyrimidine
structural that aggravate macromolecule hydrogen. The presence of the sulfonamide group leads
to increase solubility due to their effective contribution to the cohesive energy which
counteracting their influence in the increment in the main chainmain chain distance.
Inherent viscosity
The inherent viscosity ( inh ) of the polymers, as a suitable criterion for evaluation of molecular
weight, was measured at a concentration of 0.5 g/100 mL in DMSO at 30 C. The inh of
phenylene-containing polyamides 3640 were in the range 0.141.48 dL/g while their
analogues4145 were in the range 0.471.44 dL/g indicating low to moderate molecular weights
in this series. The inh of the amido- and sulfonamido-pyrimidine containing polymers 46
51, 5257, respectively, were closely similar in the range 0.241.61 dL/g. Noteworthy, no
significant change in inherent viscosity was noticed on phenylene/pyridine replacement.
FT-IR spectra
In general, the IR spectra of the phenylene-containing dinitro derivatives 812 exhibited bands in
the region 3,2533,361 cm 1 correspond to the NH str ; a characteristic band at 1,650 cm 1 due
to the C=O str amide while the NO 2 appeared at 1,541 cm 1 (NO 2asymstr ) and 1,340 cm 1 (
NO 2symstr ). The SO 2 sulfonamide in 12 appeared at 1,339 cm 1 (SO 2asymstr ), 1,158 cm 1and
(SO 2symstr ). Pyrimidine-containing dinitro derivatives 2123 and 3032 exhibited absorption
bands at 3,100 cm 1 attributed to the NH str ; a characteristic band in the region 1,626
1,685 cm 1 correspond to the C=O str amide while the NO 2 bands appeared at 1,540 cm1 .
The IR analyses of the phenylene- and pyrimidine-containing diamines 1317, 2426 and 33
35showed absorption bands in the region 3,4333,454 cm 1 correspond to the amino groups (
NH2asymstr ) and 3,3603,394 cm 1 due to (NH 2symstr ); the band at 3,250 cm 1 due to the
NH strband while the C=O str amide bands were in region 1,6401,680 cm 1 . The
SO 2 sulfonamide bands in the diamines 3335 appeared at 1,345 cm 1 (SO 2asymstr ) and
1,159 cm 1 (SO2symstr ).
The IR analyses of the phenylene-containing polymers 3640 exhibited major absorption bands,
respectively, 36: 3,275 (NH str ), 3,094 (CH str arom), 1,663 (C=O str amide), 1,600
(C=C strarom); 37: 3,387, 3,235 (NH str amide), 3,081 (CH str arom), 1,688, 1,660
(C=O str amide), 1,590 (C=C str arom), 733 (CCl str ); 38: 3,291 (NH str amide), 3,081
(CH str arom), 1,689, 1,661 (C=Ostr amide), 1,593 (C=C str ), 825 (CCl str ); 39: 3,298
(NH str amide), 3,109 (CH str arom), 1,661 (C=O str amide), 1,599 (C=C str arom), 874 (C
Cl str ); 40: 3,339 (NH str amide), 3,105 (CH strarom), 1,924, 1,665 (C=O str amide), 1,594
(C=C str arom), 1,330 (SO 2asymstr ), 1,250, 1,158 (SO 2symstr ).
The IR analyses of the pyridine-containing polymers 4145 showed major absorption bands,
respectively, 41: 3,427 (NH str amide), 2,968 (CH str arom), 1,675 (C=O str amide), 1,600
(C=C strarom), 1,328 (CN str arom); 42: 3,406 (NH str ), 3,312 (NH str amide), 1,688, 1,623
(C=O stramide), 1,591 (C=C str arom), 1,346 (CN str arom), 744 (CCl str ); 43: 3,427 (NH str ),
3,298 (NHstr amide), 3,107 (CH str arom), 1,677 (C=O str amide), 1,599 (C=C str arom), 1,310
(CN str ), 873 (CCl str ); 44: 3,728, 3,298 (NH str amide), 3,106 (CH str arom), 1,678
(C=O str amide), 1,599 (C=C str arom), 828 (CCl str ); 45: 3,266 (NH str amide), 3,105
(CH str arom), 1,682 (C=O stramide), 1,595 (C=C str arom), 1,330 (SO 2asymstr ), 1,159 (SO 2symstr ),
725 (CCl str ).
The IR spectral data of the pyrimidineamido-containing polymers 4651 exhibited absorption
bands, respectively, 46: 3,399 (NH str amide), 1,655 (C=O str amide), 1,536 (C=C str arom); 47:
3,432 (NH str amide), 1,657 (C=O str amide), 1,610 (C=C str arom); 48: 3,396 (NH str amide),
1,658 (C=O str amide), 1,536 (C=C str arom); 49: 3,438 (NH str amide), 1,696, 1,666
(C=O stramide), 1,629 (C=N str arom), 1,595 (C=C str arom); 50: 3,435 (NH str amide), 1,681
(C=O stramide), 1,607 (C=C str arom); 51: 3,444 (NH str arom), 3,294 (NH str amide), 3,093
(CH str arom), 1,695, 1,667 (C=O str amide), 1,595 (C=C str arom).
The IR spectral data of the pyrimidinesulfonamido-containing polymers 5257 showed
absorption bands, respectively, 52: 3,435 (NH str amide), 1,659 (C=O str amide), 1,597
(C=C str arom), 1,326 (SO 2asymstr ), 1,154 (SO 2symstr ); 53: 3,404 (NH str amide), 1,665
(C=O str amide), 1,596 (C=Cstr arom), 1,327 (SO 2asymstr ), 1,154 (SO 2symstr ); 54: 3,432
(NH str amide), 1,678 (C=O stramide), 1,599 (C=C str arom), 1,301 (SO 2asymstr ), 1,154
(SO 2symstr ); 55: 3,435 (NH str amide), 1,668 (C=O str amide), 1,595 (C=C str arom), 1,321
(SO 2asymstr ), 1,153 (SO 2symstr ); 56: 3,437 (NH str amide), 1,678 (C=O str amide), 1,595
(C=C str arom), 1,322 (SO 2asymstr ), 1,153 (SO2symstr ); 57: 3,440 (NH str amide), 1,680
(C=O str amide), 1,597 (C=C str arom), 1309 (SO2asymstr ), 1,151 (SO 2 asymstr ).
Optical properties
The optical properties of the polyamides 3645
The optical properties of polyamides series containing chloroaromatic pendent moiety and 36
45were investigated by UVvis and photoluminescence spectroscopy in DMSO using
concentration of 2 mg/10 ml. The values of molar extinction coefficients were in the range
14,64023,530 M 1 cm1 . The PL spectra were measured at 290 nm excitation.
wavelength using polymer concentration of 10 4 . Table 1 compiles the optical data of this
polymer series and several interesting points are concluded:
Relative to the unsubstituted polyamide, all substituted polymers showed slightly shifted
absorption peaks due to the electronic effect of the substituent that increase the electron density,
thereby leading to a relatively large energy band gap for * transitions.
The fluorescence emission spectra of all polyamides exhibited two emission peaks at 346 nm and
580 nm.
The orange emission observed for all polyamides at 580 nm could be attributed to the
substituent electronic effect.
Pyridine containing polyamide 41 exhibited slightly blue shifted absorption band relative to its
phenylene analogue 36 while its emission showed a red shifted emission peak at 420 nm.
Compared to a benzene ring, pyridine has a greater electron affinity and better electron-
transporting properties.
Table 1. The optical properties of polyamides 3645
The optical properties of pyrimidine containing polymers 4651 showed the values of molar
extinction coefficients are in the range 43,60074,100 M 1 cm 1 and the optical data are
collected in Table 2. From the UVvis spectral data given in Table 2 several remarks are found:
Pyrimidine containing polyamide 46 exhibited a blue shifted absorption peak at 267 nm relative
to its phenylene analogue 36.
Introduction of one methyl substituent led to a new absorption at 312 nm while the presence of
two methyl substituents red-shifted the peak to 331 nm.
Pyridine containing polyamides 4951 exhibited similar absorption peaks at 275 nm and no
further changes were noticed in presence of substitution. Relative to their phenylene
analogues 4648, these series showed red shifted absorption (up to 10 nm).
Green emission observed in this series at 550 nm in addition to the combined peak at 346 nm.
Table 2. Optical properties of polyamides 4651

The optical properties of pyrimidine containing polymers 5257 showed the values of molar
extinction coefficients are in the range 48,40074,100 M 1 cm 1 and the optical data are
collected in Table 3. From the UVvis spectral data several remarks are found:
All polymers in this series exhibited yellow emissions at 572 nm. No change upon
phenylene/pyridine exchange except polymer 56 in which replacement red shifted the absorption.
Furthermore, methyl substitution red-shifted the absorption bands.
This series of polyamides exhibited red-shifted absorptions and emission peaks relative to their
pyrimidine-containing polymers. This could be attributed either to the sulfonamides electronic
effect or the increase of molecular polarizability which reduce the energy level separation.
Table 3. Optical properties of polyamides 5257

Thermal analysis
Thermal properties of the polyamides 36-45
The thermal properties of the prepared polymers were evaluated by differential thermo
gravimetric (DTG) and differential thermal analysis (DTA) techniques. Thermal stability of the
polymers was studied in the range 20700 C (char yield), Table 4. Structureproperty
relationship demonstrated an interesting connection between a single structure change and its
thermal property.
Table 4. Thermoanalytical data of the polymers 3645
Phenylene-containing polymers 3640 exhibited similar degradation behavior. DTA analysis
revealed that the polyamide 36 exhibited an endothermic peak at 440 C and an exothermic peak
at 634 C. The TGA exhibited degradation processes at 158 C (8.6 % wt loss), 303 C (8.5 % wt
loss), 438 C (16.3 % wt loss) and 666 C (64.9 % wt loss) leaving 1.8 % as a mass residue. The
DTA analysis of 37 exhibited a single exothermic peak at 607 C, while its TGA analysis
exhibited degradations at 107 C (1.5 % wt loss), 197 C (3.7 % wt loss), 263 C (3.6 % wt loss),
463 C (43.6 % wt loss) and 676 C (46.9 % wt loss) leaving 0.7 % of as a residue. DTA analysis
of the polyamide 38 exhibited an exothermic peak at 588 C, while its TGA chart exhibited
degradation processes at 196 C (4.6 % wt loss), 291 C (20.7 % wt loss), 435 C (27.5 % wt
loss) and 643 C (45.5 % wt loss) leaving 1.7 % mass residue. DTA analysis of the
polyamide 39 exhibited an exothermic peak at 590 C and its TGA exhibited degradation
processes at 205 C (11.2 % wt loss), 298 C (3.3 % wt loss), 466 C (20.9 % wt loss) and
634 C (65.8 % wt loss) leaving 0 % mass residue. DTA analysis of the polyamide 40 exhibited
an exothermic peak at 607 C. The TGA exhibited degradation processes at 201 C (6.9 % wt
loss), 412 C (13.9 % wt loss) and 656 C (73.9 % wt loss) leaving 5.3 % mass residue.
Pyridine-containing polymers 4145 exhibited slightly higher thermal stability compared to their
phenylene analogues. The DTA of the polyamide 41 exhibited an endothermic decomposition
peak at 437 C and two other exothermic peaks at 480 C and 581 C, while its TGA analysis
showed successive degradation processes at 338 C (11.4 % wt loss), 489 C (30.3 % wt loss),
596 C (41.1 % wt loss) and 700 C (8.2 % wt loss), leaving 8.96 % mass residue. The
polyamide 42exhibited an exothermic decomposition peak at 609 C (DTA analysis), while TGA
exhibited degradation processes at 146 C (2.1 % wt loss), 296 C (8.2 % wt loss), 456 C
(35.6 % wt loss) and 680 C (50.0 % wt loss), leaving 4.19 % mass residue. The
polyamide 43 exhibited two exothermic decomposition peaks at 405 C and 577 C (DTA
analysis), while its TGA analysis exhibited degradation processes at 150 C (3.4 % wt loss),
287 C (7.7 % wt loss), 425 C (28.7 % wt loss) and 640 C (57.8 % wt loss), leaving 2.4 % as a
residue. The polyamide 44exhibited two exothermic decomposition peaks at 491 C and 610 C
(DTA analysis), while the TGA analysis exhibited degradation processes atv153 C (8.5 % wt
loss), 364 C (9.9 % wt loss), 503 C (30.5 % wt loss), 640 C (42.4 % wt loss) and 701 C (2 %
wt loss), leaving 6.7 % mass residue. Similarly, the polyamide 34 exhibited two exothermic
decomposition peaks at 533 C and 611 C (DTA analysis), while the TGA exhibited
loss), 621 C (28.9 % wt loss) and 670 C (3.9 % wt loss), leaving 8.1 % as a residue. Thus, the
introduction of pyridine moiety in the main chain of a polymer imparts thermal stability. The
DTG curves of some representative examples of this series are shown in Fig. 4.
Fig. 4. DTG curves of polymers 3842 and 45

Thermal properties of the polyamides 4651
Pyrimidine-containing polymers 4651 exhibited relatively high thermal stability compared to
their analogues 3645, Table 5. The major degradation occurred in the range 400700 C leaving
traces of the polymer as a mass residue. The introduction of the methyl substituent in the main
chain of a polymer has no significant effect on the thermal stability. The DTA curve of the
polyamide 46 exhibited an exothermic peak at 537 C. The TGA curve showed successive
loss) and 699 C (61.9 % wt loss), leaving 0.3 % of as a mass residue.
The polyamide 47 exhibited an endothermic peak at 592 C and another exothermic peak at
656 C (DTA analysis). The TGA showed degradation processes at 443 C (26.71 % wt loss),
550 C (19.4 % wt loss) and 700 C (52.2 % wt loss), leaving 1.8 % mass residue. The
polyamide48 exhibited an exothermic peak at 607 C (DTA analysis). The TGA analysis showed
successive degradation processes at 215 C (11.58 % wt loss), 403 C (16.6 % wt loss) and
597 C (68.5 % wt loss), leaving 3.32 % as a residue.
Pyridine-containing polyamide 49 exhibited an exothermic decomposition peak at 633 C (DTA
analysis). The TGA analysis exhibited four degradation processes at 99 C (6.9 % wt loss),
197 C (3.3 % wt loss), 398 C (24.1 % wt loss) and 631 C (65.5 % wt loss), leaving 0.23 % as
a mass residue. The DTA analysis of the polyamide 50 exhibited an exothermic peak at 555 C.
The TGA analysis showed three degradation processes at 205 C (8.5 % wt loss), 379 C
(15.2 % wt loss) and 606 C (72.6 % wt loss), leaving 3.7 % as a residue. The
polyamide 51 exhibited an exothermic peak at 574 C (DTA analysis). The TGA analysis
showed five degradation processes at 137 C (7.0 % wt loss), 213 C (2 % wt loss), 275 C
(11.7 % wt loss), 424 C (17.3 % wt loss) and 670 C (62.1 % wt loss), leaving 0 % residue.
Thermal properties of the polyamides 5257
Thermal properties of the polyamides 5257 are collected in Table 6 and the results revealed
comparable thermal stability. As indicated by the DTA chart, the polyamide 52 exhibited two
exothermic decomposition peaks at 491 C and 539 C. The DTG curve exhibited degradation
processes at 174 C (8.1 % wt loss), 385 C (34.9 % wt loss), 451 C (11.2 % wt loss) and
622 C (45.5 % wt loss), leaving 0.24 % as a residue. The polyamide 53 showed two exothermic
peaks at 533 C and 596 C (DTA analysis). The TGA exhibited degradation processes at 73 C
(5.4 % wt loss), 497 C (39.7 % wt loss) and 663 C (53.3 % wt loss), leaving 1.67 % as a mass
residue.
The polyamide 54 exhibited two exothermic peaks at 440 and 581 C (DTA analysis). The TGA
analysis showed five degradation processes at 133 C (6.1 % wt loss), 340 C (22.1 % wt loss),
450 C (21.4 % wt loss), 502 C (19.1 % wt loss) and 590 C (28.3 % wt loss), leaving 3.1 %
mass residue. The DTA analysis revealed that 55 exhibited two endothermic peak at 460 C and
584 C, while the TGA analysis exhibited degradation processes at 194 C (10.0 % wt loss),
303 C (3.5 % wt loss), 411 C (19.3 % wt loss) and 647 C (65.9 % wt loss), leaving 1.3 % of
the polymer as a mass residue. The polyamide 56 exhibited two exothermic decomposition peaks
at 563 and 589 C (DTA), while the TGA chart showed degradation processes at 160 C (7.9 %
wt loss), 392 C (16.0 % wt loss), 566 C (53.3 % wt loss) and 629 C (19.5 % wt loss), leaving
3.2 % as a remaining mass residue. The DTA data of the polyamide 57 showed an endothermic
peak at 372 C and exothermic peak at 593 C. The TGA analysis exhibited degradation
processes at 81 C (5.6 % wt loss), 410 C (18.2 % wt loss), 528 C (21.0 % wt loss), 600 C
(12.9 % wt loss) and 700 C (32.9 % wt loss), leaving 10.1 % as a mass residue. The DTG
curves of some representative examples of this series are shown in Fig. 5.
Fig. 5. DTG curves of polymers 47, 48, 51, 53, 54 and 57

In summary, pyrimidine-containing polyamides exhibited relatively higher thermal stability
compared to their sulfonamido-pyrimidine analogues. This may be explained by the feature of
the supramolecular structure, namely by a high density of packing of polymeric chains, realized
through a level-by-level stacking of these chains. With such stacking a strong intermolecular
interaction between the amide fragments of adjacent polymeric chains is provided. However, in
the case of the former polymers, the interchain interaction can occur due to specific contacts
between the amide fragments of one chain and the nitrogen atoms of the pyrimidine cycles of the
other chain. Owing to this fact the pyrimidine cycles serve as an additional amplifier of the
interchain interaction in polyamides, thus causing the strength and thermal stability to increase.
The presence of a sulfonamide group adjacent to pyrimidine, as in the case of the latter polymer
series, led to decrease thermal stability. This may be attributed to the acidic nature of the
hydrolyzable group that retain the high polarity and thus, the polymer degraded before melting
stage. Nevertheless, the methyl substitution enhanced the thermal stability in this series.
Calculations of limiting oxygen index

Flammability of polymers is one important property which could limit their applications [30].
Despite the fact that high-performance polymeric materials offer many advantages over
conventional metals, their flammability and possible release of toxic byproducts increase the fire
risk and thus the introduction of flame-retardant additives are the easiest way to diminish the
polymer flammability. The flame retardancy is evaluated by limiting oxygen index (LOI). The
LOI is defined as the minimum oxygen concentration needed in an inert gas medium for the
material to achieve burning after ignition. The LOI is a measure of the ratio of oxygen to other
gases in the air surrounding a substrate. A material with an LOI of greater than 21 % but less
than 28 % would be considered slow burning while a material with an LOI of greater than
28 % would be considered self-extinguishing. Char yield can be used as criteria for evaluating
LOI of the polymers in accordance with Van Krevelen and Hoftyzer equation [31];
LOI = 17 + 0.4CR, where CR = chars yield. The calculated LOI values of all polymers based on
their char yield were less than 28, Tables 7,8.
Table 7. The char yields and LOI values of the polyamides 3645
Table 8. The char yields and LOI values of the polyamides 4657
Calculations of thermodynamic parameters
The thermodynamic parameters of decomposition processes of polymers, namely, activation
energy E enthalpy (H), entropy (S) were evaluated by employing the Horowitz-Metzger
equation [32], Additional file 1: Tables S1, S2. The order of chemical reactions (n) was
calculated via the peak symmetry method by Kissinger [33]. The asymmetry of the peak, S, is
calculated as follows:
(1)
(2)
The value of the decomposed substance fraction, m, at the moment of maximum development
of reaction (with T = T m ) being determined from the relation (3):
(3)
The values of collision factor, Z, can be obtained in case of Horowitz Metzger by making the use
of the relation (4):
(4)
where S is the entropies of activation, R represents molar gas constant, rate of heating (K s 1),
K the Boltzmann constant, and h the Plancks constant [34]. The change in enthalpy (H) for any
phase transformation taking place at any peak temperature, Tm, can be given by the following
equation: S = H/Tm. Based on least square calculations, the Ln T versus 1,000/T plots for
all complexes, for each DTA curve, gave straight lines from which the activation energies were
calculated according to the reported methods [35]. The slope is of Arrhenius type and equals
E/R.
The kinetic data obtained from the nonisothermal decomposition of the prepared polyamides
series containing chloroaromatic pendent moiety 3645 are given in Additional file 1: Table S1.
The following trends and conclusions may be achieved:
1. The calculated values of the collision number, Z, showed a direct relation to Ea. The
maximum and minimum Z values for polyamides 3640 derived from isophthaloyl dichloride
were 7.28 S 1and 1.014 S 1 and that derived from pyridine 2,6-dicarbonyl dichloride 41
45 were 12.69 S 1and 1.02 S 1 suggesting different degradation mechanisms with variable
speeds. The values of the decomposed substance fraction, m for the polyamides 3640 at the
maximum development of the reaction are of nearly the same magnitude and lie within the range
0.480.64.
2. The change of entropy values, S, for all polymers has nearly the same magnitude lie within
the range 0.23 to 0.25 kJ K 1 mol 1 and the negative signs of the entropy suggest ordered
transition states, i.e., in a less random molecular configuration. The fractions appeared in the
calculated order of the thermal reactions, n, confirmed that the reactions proceeded in
complicated mechanisms.
3. Activation energies (E) of polyamides 3640 demonstrated lower values compared to their
partners 4145. The first and second decomposition steps in some polymers have nearly equal
E values, indicating similar degradation mechanism.
4. The enthalpy (H) of polyamides 3640 demonstrated higher values compared to their
partners 4145, respectively, and the negative values demonstrated the exothermic
decomposition processes.
The kinetic data obtained from the nonisothermal decomposition of the polymers 4657 are
given in Additional file 1: Table S2. The following trends and conclusions may be achieved:
1. The maximum and minimum collision number Z values for polyamides 4650 are 3.45 and
0.93 S 1 , respectively, while that for the polyamides 5157 are 26.6 and 1.03 S 1 suggesting
different degradation mechanisms with variable speeds. Noteworthy, the collision number Z
values for polyamides containing sulfonamide group are 1.57, 1.2, 0.9, respectively, suggesting
similar degradation mechanisms. The values of the decomposed substance fraction, m for
polyamides at the maximum development of the reaction are of nearly the same magnitude and
lie within the range 0.330.73.
2. The entropy values, S, for all polymers have nearly the same magnitude and were in the
range 0.22 to 0.25 kJ K 1 mol 1 . The observed negative signs clearly demonstrated that the
transition states are more ordered, i.e., in a less random molecular configuration. The fractions
appeared in the calculated order of the thermal reactions, n, confirmed that the reactions
proceeded in complicated mechanisms.
3. Activation energies (E) of polyamides 4650 demonstrated lower values compared to their
partners 5157. Noteworthy, polyamides containing sulfonamide exhibited higher E and methyl
substitution produced polymer have high E than their unsubstituted analogs.
4. The enthalpy (H) of polyamides 4650 demonstrated higher values compared to their
partners 5157 and the negative values demonstrated exothermic decomposition processes.
Biological properties
Antimicrobial activity
The antimicrobial activity of the polyamides series 3657 were examined against a variety of
microorganisms included fungi such as: A. fumigatus RCMB 02568, S. racemsum RCMB
05922, G. candidum RCMB 05097 and C. albicans RCMB 05036; gram positive bacteria such
as: S. pneumoniae RCMB 010010 and B. subtilis RCMB 010067; and gram negative bacteria
such as: P. aeruginosa RCMB 010043 and E. coli RCMB 010052. In all cases, the diffusion agar
technique was applied and the antimicrobial activity results are collected in Additional file 1:
Tables S3S5.
Antimicrobial activity of polymeric series 3645
Chloro aromatic compounds played a vital role in the development of different medicinal agents
where chlorine is electronegative, and therefore oxidizes peptide link and denatures proteins.
Exposure of strains of E. coli, Pseudomonas spp. and Staphylococcus spp. to lethal doses causes
a decrease in ATP production. Chlorine acts on the permeability of the external membrane of E.
colithrough a primary lethal phenomenon which consists in a substantial leakage of K + ions;
such leakage does not occur for macromolecules. Sub-lethal doses inhibit cellular respiration due
to a nonspecific oxidizing effect (bactericidal effect) [36].
Results of antimicrobial activity of polyamides 3640, derived from isophthaloyl chloride, and
the comparative activity of currently used antibacterial and antifungal agents are presented in
Additional file 1: Table S3. Thus, the introduction of choro substituents clearly enhanced the
antimicrobial activity against all fungi and B. subtilis with inhibition zone diameters ranging
between 13.4 and 19.6 mm. Compared to other analogues, the polyamide containing p-chloro
substituent showed higher activity against the tested microorganisms.
The antimicrobial activity comparative tests of the polymeric series 4145, derived from
pyridine 2,6-dicarbonyl dichloride, are presented in Additional file 1; Table S4. Polyamides
containing both sulfonamide and chloro substituents showed higher antimicrobial activity against
all fungi and gram positive bacteria. The presence of such bioactive groups in the backbone of
the polymer play the key role in catalyzing both biological and chemical systems. Compared to
their analogues 2529, the polyamides 3034 showed relatively higher antibacterial activities
against all tested microorganisms.
Antimicrobial activity of polymeric series 4657
Sulfonamides are chemotherapeutic agents which display various biological interactions,
including inhibition of carbonic anhydrase and affecting insulin releasing in addition to their
antimicrobial, antitumor and anti-inflammatory activities. The antimicrobial activity of the
amido- and sulfonamido-pyrimidine containing polymers 4657 are presented in Additional
file 1: Tables S4, S5. From the screening results, the following remarks are concluded:
Pyrimidine-containing polyamides exhibited high antifungal activity than their analogues
containing sulfonamidopyrimidine pendant structures. Thus, the presence of sulfonamide
structures in such polymeric series considerably alters the antimicrobial activity of the polymer.
The polyamides4648 exhibited remarkable antifungal activities against A. fumigatus and,
interestingly, the observed activity were more potent than those of the reference Amphotericin B.
Over 80 % of the reported Aspergillus-related cases, such as extrinsic allergic alveolitis, asthma,
allergic sinusitis, chronic eosinophilic pneumonia, hypersensitivity pneumonitis, and allergic
bronchopulmonary aspergillosis are most frequently caused by A. fumigatus[37].
Moreover, introduction of methyl substituents in case of the polyamides 47 and 48 produced
potent antifungal polymers against S. racemosum and, the noteworthy, the activity were higher
than the reference Amphotericin B and thus, the introduction of a methyl group to the pyrimidine
promotes antifungal activity. S. Racemosum is well known to cause skin and soft tissue infection
and fungal rhinosinusitis [38].
Sulfonamidopyrimidine-containing polyamides analogues 5153 exhibited higher antibacterial
activity against gram negative bacteria than their analogues 4648. Thus, replacement of the
amide linkage by sulfonamide linkage promoted specifically the antibacterial activity against
gram negative type. Noteworthy, relative to the reference antibiotic Gentamicin, the
polyamide 54exhibited comparable antibacterial activity against gram negative bacteria. It has
been reported that P. aeruginosa is the most common pathogen causing chronic infection in
people with cystic fibrosis (an inherited disease that affects the lungs, digestive system and sweat
glands) [39].
Pyrimidine-containing polymer analogue 52 exhibited remarkable antibacterial activities
against S. pneumoniae, a gram positive bacterium and P. aeruginosa a gram negative bacterium.
In both cases, activities were more potent compared to the references antibiotics used. Thus, the
polyamide 52 is likely to be a promising broad spectrum antibacterial agent.
In general, polymers have pyrimidinoamide linkages exhibited lower activities toward gram
positive bacteria than their analogues have sulfonamidopyrimidine linkage.
The polyamide analogue 57 exhibited promising antibacterial activities against both gram
positive and gram negative bacteria. Interestingly, its activity as reflected by the inhibition zone
diameter is higher than the reference antibiotic Gentamicin.
Antimicrobial activities statistical analyses
The antimicrobial activity data of the most promising polymers were analyzed against their
corresponding controls using SPSS software package version 18.0 (SPSS, Chicago, IL, USA).
These included the cases at which the structures 3537, 38, 40, 43 and 46 exceeded the activities
of the examined reference antimicrobial agents. Quantitative data were analyzed using an F-test
and the results are presented in Additional file 1: Table S7 and Fig. 6. The p value was assumed
to be significant at 0.05. From the screening results, the following remarks are concluded:
A. fumigatus was significantly (p < 0.001) sensitive to the tested pyrimidine-containing
polymers4648 compared to the control.
S. Racemosum was significantly (p < 0.003) sensitive to substituted pyrimidine-containing
polymers 4748 compared to the control.
B. subtilis was significantly (p < 0.004) sensitive to the polymer 50 compared to the
analogues 49and 51 and the control.
S. pneumonia was significantly (p < 0.001) sensitive to the polymer 52 which exhibited broad
antibiotic spectrum compared to the analogues 5354 and the control.
Fig. 6. Inhibition zone values of the potent polymers 4655, 57 and the standard
against fungi: A. fumigatus, S. racemsum, G. candidum and C. albicans; and bacteria; gram
positive bacteria: S. pneumoniae and B. subtilis; and gram negative bacteria: P.
aeruginosa and E. coli.
Although these polymers have shown remarkable antimicrobial activity, further studies need to
be conducted to ascertain the exact mechanism of the activity and the minimal inhibitory
concentration.
Conclusions
A series of aromatic polyamides containing substituted halogenated aromatic, pyrimidineamido-

and pyrimidinesulfonamido pendent structures in bulk and nanoscale were synthesized and
screened for their antimicrobial activity against microorganisms. The SEM analysis of
polyamides indicated that most of them were organized as well defined nano sized spheres but in
case of pyridine and pyrimidine containing polyamides small amount of aggregated nanospheres
were also observed. Thermal analysis of the polymers was studied in the temperature range 20
700 C and results showed comparable thermal behavior.
The optical results showed that polyamides series containing chloroaromatic pendent moiety
exhibited orange emission at 580 nm. The pyrimidineamido polymeric series showed green
emission 550 nm while their pyrimidinesulfonamido analogues exhibited yellow emission
572 nm in addition to the blue emission at 482 nm. Interestingly, structural modification via
benzene/pyridine interchange resulted in red shifted emission peaks in most cases and this could
be attributed to the localized lone pair of electrons in the sp 2 orbital of the nitrogen atom which
offer the polyamide a greater electron affinity and better electron-transporting properties.
Biological results showed that the halogenated polyamides exhibited good antimicrobial activity
against most tested microorganisms. The amido- and sulfonamidopyrimidine containing
polymers exhibited most potent antimicrobial agents in the present series. Polymers having
pyrimidinoamide linkages exhibited lower activities toward gram positive bacteria than their
analogues have sulfonamidopyrimidine linkage. Relative to the reference antibiotic Gentamicin,
the polyamide 54exhibited comparable antibacterial activity against gram negative bacteria (P.
aeruginosa); the most common pathogen causing chronic infection in people with cystic fibrosis.
Pyrimidine-containing polymer analogues 52 and 57 exhibited remarkable antibacterial activities
against gram positive and gram negative bacteria. In both cases, activities were more potent
compared to the references antibiotics used. Thus, these polyamides are likely to be promising
broad spectrum antibacterial agents and deserve further investigation in order to clarify the mode
of action at the molecular level.
Additional file
Additional file 1:. Table S1. Kinetic parameters of the polymers 3645. Table S2. Kinetic
parameters of the polymers 4657. Table S3. Antimicrobial activity of polyamides 2529(discs
6 mm). Table S4. Antimicrobial activity of polyamides 4145 (discs 6 mm). Table
S5. Antimicrobial activity of polyamides 4651 (discs 6 mm). Table S6. Antimicrobial
activity of polyamides 5257 (discs 6 mm). Table S7. Statistical analysis of some polyamides
exhibited promising antimicrobial agents.
Format: DOCX Size: 51KB Download file
HHAMH, ESMEM designed the research point, monitoring the progress of the chemistry work,
analyzed and write the manuscript. AMSAZ carried out the preparation of the monomers and
polymers. AFE analyzed and write the thermal data. ERE and RS analyzed and write the
biological section. All authors read and approved the final manuscript.
The article part of MSC thesis of AMSAZ.
Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.
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Absorptive stripping voltammetry for cannabis detection

Department of Chemistry, Physical & Theoretical Chemistry Laboratory, Oxford University,

Received: 27 March 2015

9 -tetrahydrocannabinol (THC), the active constituent of cannabis, is known to reduce

Fig. 1. a Square wave voltammograms (frequency: 100 Hz, amplitude: 40 mV,

Fig. 2. a Square wave voltammograms (frequency: 100 Hz, amplitude: 40 mV,

Fig. 3. a Square wave voltammograms (frequency: 100 Hz, amplitude: 40 mV,

Fig. 4. a Square wave voltammograms (frequency: 100 Hz, amplitude: 40 mV,

The authors declare that they have no competing interests.

For all author emails, please log on.

Received: 3 January 2015

Accepted: 10 July 2015

Published: 9 September 2015

2015 Enrquez-Nez et al.

Fig. 1. The ping-pong bi-bi scheme mechanism

Results and discussion

Fig. 3. Chromatogram of the end of the enantioselective hydrolysis. (S)-

malathion was determined to be in 96%ethanol, indicating an ee of at least

Fig. 5. Chromatogram of the isolated (S)-malathion monocarboxylic acid. (S)-

Fig. 6. Racemic malathion monocarboxylic acid. (S)-malathion monocarboxylic

Fig. 7. 1 H NMR spectrum of malathion

Enantioselectivity value (E-value) measurements

Isolation of (R)-malathion and racemization of (S)-malathion monocarboxylic acid

Comparison of commercially available lipases as biocatalysts for the enantioselective hydrolysis

The authors declare that they have no competing interests.

1. Potec I, Cieslak L, Sledzinski B, Ksycinska H. Simple synthesis of malathion and

Received: 5 January 2015

Accepted: 10 July 2015

Published: 27 August 2015

2015 Vasylechko et al.

Reagents and apparatus

Results and discussion

Fig. 2. TG and DTG-curves of natural clinoptilolite (1, 1*) and clinoptilolite

Fig. 3. DTG-curves of Transcarpathian clinoptilolite previously heated at

A solid-phase extraction procedure was developed for spectrophotometric determination of trace

The authors declare that they have no competing interests.

1. Emsley J. The elements 3-rd edition. Clarendon Press, Oxford; 1998.

Received: 6 March 2015

Accepted: 16 July 2015

Published: 7 August 2015

2015 Lavakumar et al.

Results and discussion

UVVis spectral analysis

Fig. 1. Formation of silver nanoparticles by green synthesis. AgNPs were

Fig. 2. UV-Vis absorption maxima of silver nanoparticles. The data is based on

Fig. 3. Particles size distribution of AgNPs prepared from G. crassaextracts.

Fig. 4. Zeta potential illustration of AgNPs. Zeta potential distribution of

Fig. 5. X-ray diffraction analysis of bio synthesized silver nanoparticles using

Fig. 6. Morphological images of synthesized AgNPs by using FESEM. This

Fig. 8. Antibacterial activity of AgNPs. Zone of inhibition of silver

Fig. 9. Sensitivity prototypes of silver nanoparticles against various microbial

FESEM: field emission scanning electron microscopy

EDAX: energy dispersive analysis of X-rays

XRD: X-ray diffraction

Fcc: face centered cubic

SPR: surface plasmon resonance

ROS: reactive oxygen species

MTCC: microbial type culture and collection

Compliance with ethical guidelines

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Received: 27 March 2015

Accepted: 19 June 2015

Published: 1 July 2015