PKa Modulation in rhodamine based probes for colorimetric detection of picric acid

Article abstract of DOI:10.1039/c4ob02001d

Tuning the pK_a in acid sensitive rhodamine spirolactam derivatives as a function of the solvent medium resulted in the selective detection of picric acid from its lower nitro phenolic analogues and a few other carboxylic acids. This journal is

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DOI: 10.1039/C4OB02001D
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ARTICLE TYPE
pKa Modulation in Rhodamine Based Probes for Colorimetric Detection
of Picric Acid
V. Nagarajana and Bamaprasad Baga
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Tuning of pKa in acid sensitive rhodamine spirolactam 45 Picric acid (PA) is an explosive material of nitro aromatics family
derivatives as a function of solvent medium resulted in
selective detection of picric acid from its lower nitro phenolic
analogues and few other carboxylic acids.
that also have been utilized for various applications3, however,
causes mild to severe damage on human health depending upon
the intake. Thus, their detection in traces is highly desirable in
environmental perspective. Although various efficient materials
50 and methods are known for their quantifiable detection,
colorimetric/fluorometric probes have perceived much attention
recently due to their high sensitivity and real time monitoring.
Such probes are categorized into two groups based on their
interaction with PA through (a) the electron deficient phenyl
55 ring4 and (b) the acidic hydroxyl group.5 The former type of
probes, however, lack in good selectivity if PA is part of a mixed
analyte containing other nitro aromatics, because the donor-
acceptor complex formation is feasible too for all nitro aromatics.
The latter type show high selectivity for PA among nitro aromatic
60 explosives as those interfering entities generally do not have the
acidic hydroxyl group. However, their selectivity gets severely
affected if other sensitive acidic components present in the mixed
analyte. For example, degradation of PA lead to possible acidic
products such as dinitrophenol, nitrophenol, phenol, etc,6 which
65 may affect the selective detection of PA. Thus, probes that
selectively identify PA from both nitro aromatics and other acidic
10 The contrast colour change due to structural transformation of
organic molecules in colligation with pH is promising mode for
selective detection of various acidic/alkaline analytes. Rhodamine
spirolactam derivatives (RSL), a class of xanthene dyes, being
such acid responsive probes are suitable for such prototype
15 approach. These derivatives are also known for their usage in
detection of various metal ions1 relying on a structure-function
correlation. The analyte triggers a structural transformation in
RSLs from their colourless spirolactam conformation to a
conjugated pink coloured ring-opened amide form to result in a
20 signal modulation which can be visually perceived as well as
instrumentally monitored. Although RSLs have shown to
recognize the acidic environment of different systems in various
studies,2 selective detection of an organic acid from a solution
containing mixture of its possible degraded acidic analogues, to
25 the best of our knowledge, is not explicitly known. The acid
mediated ring opening of spirolactam form of an RSL depends
upon strength of acidic entity measured in pKa. It is apparent that
pKa value of an acid varies if non-aqueous solvent is employed
as a co-solvent along with water, because of its varied
30 dissociation ability in different solvent medium. Concurrently,
most of RSL derivatives have poor solubility in water, thus, the
sensing ability of acids with RSL would vary with solvent
medium. Strong acids such as mineral acids could open the
spirolactam ring elegantly irrespective of the solvent medium due
35 to their much lower pKa values as compared to RSLs. The
selectivity among them may possibly not be induced as pKa of
RSLs are not in the match scale with those of mineral acids.
Therefore, considering wide spectrum of pKa values of many
organic acids and strength relative to RSLs, their selective
40 detection is possible, in principle, based on their different
dissociation ability in different solvent medium. Herein we report
selective detection of such an organic acid, picric acid (PA), from
a mixture of nitro phenols and carboxylic acids through pKa
compatibility approach.
components are certainly advantageous. In
a
different
methodological approach, we have reasoned to choose RSL
derivatives, as they are inactive towards nitro aromatic
70 compounds and anticipated to detect PA selectively from other
acidic analytes based on varied dissociation ability of the acids in
different solvent medium.
Scheme 1
O
O
N
N
N
N
O
N
N
O
N
1
2
75 Two simple RSLs, 1, the Schiff base and 2, the alkyl derivative,
were synthesized by two and one steps reaction respectively for
the investigation. 1 was obtained by synthesizing rhodamine B
hydrazide followed by the reaction with formaldehyde and 2 was
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DOI: 10.1039/C4OB02001D
other words, any acid whose pKa is lower than that of RSLs
should, in principle, be compatible for its proton-mediated
spirolactam ring opening to exhibit spectroscopic signalling
responses as well as structural transformation induced colour
55 change. The analyte being PA here, a strong acid with a pKa
value of 0.38 in aqueous medium (ESI, Table ST1), has been
anticipated to be selectively detected from other acidic analytes of
moderate strength by using a non-aqueous solvent medium. Thus,
all experiments were carried out in acetone, a non-aqueous
60 medium, which is already known to be used as a processing
solvent for the detection of PA due to high solubility of PA.10 In
addition to 1, pH titration of 2 at varied percentage of water in
water/acetone solvent medium have also exhibited similar trend
of increase in the pKa (Fig. 1b). The extrapolated pKa range for 2
65 was estimated to be from 1.41 to 5.66. Notably, generation of
pink colour after addition of the acid during pH measurement was
observed to be instantaneous for 1, however followed a slow
kinetics for 2.
obtained by refluxing rhodamine B hydrochloride with excess
ethylamine in ethanol. The molecular ion peaks at 469.3 and
470.4 (m/z)+ in the positive ion ESI-MS spectra inferred to
formation of 1 (C29H32N4O2) and 2 (C30H35N3O2) respectively.
The unique chemical shift in their 13C NMR spectrum, at 64.27
ppm for 1 and 64.88 ppm for 2 in CDCl3 corresponding7 to a
quaternary carbon(C9) atom, has confirmed their existence in
spirocyclic conformation.
5
If the pKa of acids is variable in different solvent medium, the
10 pKa of RSL, for ring opening process mediated by that acid,
should also be variable in such conditions. Thus, to investigate
the variation in pKa values of 1 in water/non-aqueous solvent
mixture of varied compositions, a series of pH titrations were
carried out in water/acetone mixture by varying the amount of
15 water from 15-60% in the 10µM solution of 1. The absorbance at
558 nm is measured against pH range from 2-10 for each mixture,
and the pKa values obtained8 from each experiment is plotted
against the percentage of water as shown in Fig.1a.
Considering acetone as a solvent medium based on pH titration
70 experiments explained above, evaluation of signalling responses
of 1 with PA was assessed by differentiating UV-Vis absorption
spectra of 1 and its mixture with PA in acetone. The colourless
solution of
1 confirmed its existence in near exclusive
spirolactam conformation, which remained stable for several
75 days. However, addition of PA to 1 exhibited an instantaneous
pink colouration in resultant solution, attributed to ring opening
of its spirolactam form through a proton transfer from the Lewis
acid (PA) as depicted in Scheme 2.
20 Fig. 1. Plot of water percentage versus the pKa values of (a) 1 and (b) 2.
A linear increase in pKa values of 1 with respect to increase in
the percentage of water is observed within the range of 15-60%
of water in water/acetone mixture. The extrapolation of straight
line towards zero and 100% of water gave pKa of lowest of 1.89
25 and highest of 6.19 respectively. Thus, it is presumed to be
convenient to tune pKa of RSL derivatives by simply varying the
solvent medium. pKa variation with respect to substituents of
different bulkiness in spirocyclic nitrogen is known.9 Further, it is
apparent that the observed increase in pKa values of 1 due to
30 increase in percentage of water in the water/acetone binary
mixture medium, has a reverse effect with respect to the trend
would be observed for the pKa values of acids in such conditions.
Thus, it is not straight forward to correlate the pKa values of
acids with RSL for ring opening process. However, a qualitative
35 idea could be drawn from the pKa window of RSL for selective
detection of an acid by choosing an appropriate solvent system
for a particular acidic analyte. The reverse trend of pKa values of
RSLs and the acidic analyte in a particular range facilitates a
match in their respective pKa in that suitable medium which in
40 turn, induce opening of the spiro-ring of RSLs. Within a pKa
match scale for a RSL-organic acid couple therefore, a strong
acid with higher dissociation abilities could be discriminated
from other acids comparatively of moderate strengths in a solvent
medium where the percentage of non-aqueous solvent is
45 exclusive or near exclusive in aqueous medium. This is because
such condition does not favour the dissociation of relatively
weaker acids. However, increase in polarity of solvent medium
may also facilitates those organic acids with moderate
dissociation constant to open spirolactam ring of RSL along with
50 the acids of comparatively higher strength in that pKa range. In
80 Scheme 2
OH
O2N
NO2
O
H
O
N
N
N
O2N
NO2
N
N
O
NO2
N
O
N
O
N
NO2
Pink
Colorless
As anticipated, the absorption spectra of 1(25µM) in 450-650 nm
range showed no appreciable absorption (Fig. 2a). Addition of
PA to 1 immediately exhibited a strong peak with maximum of
85 558 nm (ε1+PA =26760 dm3mol−1cm−1) indicating the opening of
its spirolactam ring. However, addition of PA to the colourless
solution of alkyl derivative 2(25µM) in acetone did not result in
such instantaneous colour change, instead, the pink colour
generated slowly and saturated after 12h (Fig. 2b) along with
90 absorption enhancement at 558nm (ε2+PA =16788 dm3mol−1cm−1).
95
Fig. 2. Absorption spectra of (a) 1 and 1+PA (b) 2 and 2+ PA in acetone.
Therefore, the absorbance measurements for 2 with PA were
carried out here onwards after 12h of addition. Such slow
2
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generation of pink colour supports the slow reaction rate(ESI, 50 carried out using a 10 µM solution of 1 or 2 in acetone added
Fig. S3) in 2 for ring opening process assessed during pH titration
experiments. The reactions of 1 and 2 with PA follow first order
kinetics where the rate is much faster for 1 with k > 0.4643 s−1 in
comparison to that of 2.77×10−5 s−1 for 2. The rate constants
with a solution(20-30 µM) of phenol, 4-nitrophenol, different
isomers of dinitrophenol, and carboxylic acids such as oxalic,
malonic, succinic, glutaric, adipic, benzoic, 4-nitrobenzoic, 4-
hydroxybenzoic, 4-aminobenzoic, acetic, trimesic acids in
5
obtained therefore supported 1 to be an appropriate probe for 55 acetone. No appreciable absorption transition in 450- 650 nm
rapid detection of PA. The NMR and ESI-MS spectral analyses
of the probes with PA have further confirmed the formation of
1+PA and 2+PA complexes and support their spirocyclic ring-
window was observed for either 1 or 2 with any of these acids
utilized here, indicating the inability of the acidic analytes to
open the spirolactam ring (Fig. 4). It is interesting to note that the
degraded acidic products of PA such as phenol, 4-nitophenol and
10 opening as observed through their absorption at 558nm upon
equimolar PA addition. The quaternary carbon(C9) peaks 60 dinitrophenols with higher pKa values than PA itself, minimum
corresponding to spirocyclic conformation, which appeared at
66.04 and 64.73 in acetone-d6 for 1 and 2 in their 13C NMR
spectrum respectively, were not observed when PA was added.
15 This indicated their structural transformation to ring-opened
of 3.97 for 2,6-dinitrophenol in aqueous medium, have also failed
to render spectroscopic signals and colour change mediated by
the ring-opening process. Therefore, these probes suitable enough
to detect PA selectivity among its possible degraded nitro
conformation in their picrate complexes, which is too supported 65 phenols and some other carboxylic acids.
by their ESI-MS spectral pattern. The stoichiometric ratio of the
complex formed was evaluated from the absorption spectral
pattern using continuous variation method (Job’s plot), which
20 indicated to the formation of 1:1 molecular complex of 1 and 2
with PA (ESI, Fig. S4).
In the process of selective detection of PA with 1 and 2, influence
of various acidic analytes were assessed by measuring absorbance
at 558 nm for individual mixtures containing the acid, 1 or 2 and
PA. Addition of PA (20 µM) to the solution containing either 1
70 or 2 and any of these acids exhibited a strong absorption peak
(A558) to a comparable extent of that observed for these probes in
presence of PA only. On contrary, none or negligible changes in
absorption intensities of 1+PA and 2+PA(ESI, Fig. S7) were
observed upon addition of those interfering analytes, which
75 ascertained that the detection of PA is not affected in presence of
these acidic products investigated here. In addition, the formation
of the complexes of 1 and 2 with PA was found to be reversible
as the pink colour disappeared along with lowering of A558
absorption almost to an extent to that of analyte free probe upon
80 subsequent addition of triethylamine. Further addition of excess
of PA facilitated the absorption transition at 558nm and
regenerated the pink colour again, thus, establishes the probes’
reusability.
In general, RSLs are utilized as a dual mode probe for analyte
85 detection. However, fluorescence experiments exhibiting spectral
maxima at 580nm pertaining to PA mediated ring opening of 1
and 2 did not show notable higher sensitivity; only 2µM solution
of PA could be detected with 1 and 2 which is two-fold less than
that in colorimetric mode. Considering operational module vis-à-
90 vis sensitivity of detection and with the advantage of naked eye
detection, the colorimetric mode for detection of PA is thus
preferred here.
Fig. 3. Absorption spectral pattern of (a) 1(30µM) and (b) 2 (40µM) upon
addition of PA (0-60µM) in acetone medium.
25 The UV-Vis absorption titration of 1 and 2 with PA showed a
steady increase in the peak intensity with respect to increased
concentration of PA (Fig. 3). The equilibrium constants thus
obtained for 1 and 2 were estimated to be 3.458 ×104 M−1 and
1.731×104 M−1 respectively which inferred to the formation of
30 moderately stable complexes (ESI, Fig. S5). Further, 1(10µM)
and 2(10µM) could detect as low as 4µM concentration of picric
acid in acetone where the change in colour could be observable
through naked eye (ESI, Fig. S6).
35
In summary, the RSL derivatives 1 and 2 are identified as a
95 reversible probes for selective detection of PA among its
degraded acidic analogues. Although both 1 and 2 could detect
the lower limit of 4 µM of PA through naked eye/colorimetric
mode, 1 was observed to be promising for real time monitoring in
comparison to 2 as the former derivative showed a faster response
100 time. A qualitative idea from the pKa window of 1 and 2 obtained
due to different solvent medium helped to discriminate PA from
various acidic analytes including nitro phenols and some
carboxylic acids utilizing acetone as a non-aqueous solvent
medium that did not favour for other acidic analytes to open the
105 spirolactam ring. It may be argued that other stronger acids such
as sulfonic derivates with much lower pKa values than that of
these RSLs should facilitate the ring-opening process to render
40
Fig. 4. Absorption spectra of the mixtures of (a) 1(10µM) and (b)
2(10µM) alone and in presence of various analytes such as PA, other
45 nitrophenols and few carboxylic acids. [analytes] = 20-30 µM, acetone.
In order to evaluate the colorimetric signalling responses of 1 and
2 with different acidic analytes including the nitrophenolic
derivatives, the UV-Vis absorption spectral measurements were
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colorimetric signals; however, 1 and 2 could be demonstrated
here to selectively detect PA among its other nitrophenolic
analogues considering its utility and mechanism of its acidic
degradation and the pKa compatibility factor. Based on pKa
compatibility of RSLs, modulation of their stereo-electronic
environment and subsequent tuning as a function of solvent
medium, probes for selective detection of other organic acids are
envisaged to be developed and we are currently working along
this direction.
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BPB wishes to thank to the Director, CSIR-IMMT for financial
support through CSIR-IMMT-ESC-401 and a research associate
fellowship to VN; to CIF at Department of Chemical Sciences,
NISER, Bhubaneswar and SAIF at CSIR-CDRI, Lucknow for
8
15 respective NMR and ESI-MS spectral analyses.
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a Department of Colloids and Materials Chemistry, CSIR-Institute of
Minerals and Materials Technology, P. O.: R.R.L.; Bhubaneswar; E-mail:
bpbag@immt.res.in. Fax: +91 674 258 1637, Tel: +91 674 237 9254.
20
†Electronic Supplementary Information (ESI) available: Experimental
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titrations, equilibrium constant determination, other photo-physical, ESI-
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