AN ELECTROANALYTICAL STUDY OF THE
DRUG PROFLAVINE
Stella Th. Girousi*, Despina K. Alexiadou, Andrea K. Ioannou,
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University,
Thessaloniki 54124, Greece
Abstract
A new method for the analysis of proflavine was developed. Electrochemical behaviour of
Proflavine (PF) was investigated by cyclic (CV) and square wave voltammetry (SWV) at the
hanging mercury drop electrode (HMDE) and the carbon paste electrode (CPE). Different
parameters were tested to optimize the conditions for the determination. Optimum results
were obtained by adsorptive stripping square wave voltammetry (AdS-SWV) using CPE. Two
oxidation peaks at 0.2 and 1.0 V appeared during the anodic scan (Ebegin = -0.1 V, Eend = 1.2
V, Estep = 0.005 V, Epulse = 0.025 V, frequency = 25 Hz). The first peak is concentration and
deposition dependent. Linearity for this peak was observed in the range of (0.2 – 23.4)  10-8
M (r = 0.998). The second peak is not deposition dependent. During the cathodic scan (Ebegin =
1.2 V, Eend = -0.1 V, Estep = 0.005 V, Epulse = 0.025 V, frequency = 25 Hz) a reduction peak
appeared at 0.2 V proving the reversibility of this action. The linearity of this peak was
observed in the range of (1.17 – 117)  10-8 M (r = 0.999). In both cases saturation of the
electrode surface was observed at higher concentrations.
* Stella Th. Girousi. E-mail: girousi@chem.auth.gr
Keywords: proflavine, square wave voltammetry, adsorptive stripping voltammetry,
carbon paste electrode, hanging mercury drop electrode
1
Introduction
Historical facts show that the drug family of acridines was discovered in 1870 when
Graebe and Caro isolated a substance from the high boiling fraction of coal tar and designated
it "acridine" or acrid substance, due to the irritating effects of its vapor on the mucus
membranes [1].
Acridines are used as bactericidal, antiseptic, inhibitory and genetically active agents.
Some are also used as analytical reagents for gravimetric and titrimetric determinations of
metal cations (Ta (V), V (V), Cr (VI), Mo (VI), Au (III)) and inorganic anions (iodide,
thiosulphate, sulphate) [2], as acid–base indicators or even as colour-development reagents for
spectrophotometric and fluorimetric determinations [3,4], photokinetic indicators [5],
luminescence sensors or reagents for drug analysis [6]. The most important property of
acridines is their chemotherapeutical action [7].
Therefore, the interaction of acridine derivatives with DNA has been the subject of
considerable research. [8]. The nature of the binding of acridine derivatives with DNA has
been explained by Peacocke and Skerret. According to them acridine dyes bind doublestranded DNA by two different types of interaction. One, a strong binding process, becomes
saturated when one dye molecule is bound per 4-5 nucleotides. The other is a weak binding
mechanism which occurs at high ratios of dye to DNA, and its limit is reached when one dye
molecule is bound per nucleotide [9].
Proflavine (PF) (Fig. 1) is a heterotricyclic dye of the family of aminoacridines. It has
several uses in human medicine: the phototherapy of recurrent herpetic infections and as a
topical antiseptic agent. PF binds with DNA in a complicated manner, involving at least two
equilibrium binding constants and more than one rate of association [10]. The stronger
interaction has been identified as intercalation of the dye chromophore between the base pairs,
with consequent extension and untwisting of the double helix. Ramstein et al. [11] and
Georghiou [12] found that PF formed molecular complexes with nucleotides in aqueous
solutions and the optical properties of these complexes were studied. Complex formation
between PF and nucleosides was indicated by the absorption and fluorescence properties of
the dye [13, 14]. PF was investigated electrochemically by cyclic voltammetry (CV ) to obtain
the transfer potential as a function of the aqueous phase pH in order to assess both neutral and
ionic partition coefficients for the study of its membrane activity [15]. The interaction of
2
proflavine with herring sperm DNA has been investigated by cyclic voltammetry and UV-Vis
spectroscopy as well as viscosity measurements [16].
The objective of the work presented in this paper is to investigate PF
electrochemically with various techniques. This paper is concerned with the behaviour of the
drug in cyclic and square-wave polarography using hanging mercury drop electrode (HMDE),
as well as with cyclic and square-wave voltammetry using carbon paste electrode (CPE).
Experimental
Reagents
All solutions were prepared from analytical reagent grade materials using doubly-distilled
water. Stock solutions of Proflavine (Aldrich, www.sigmaaldrich.com) 10-3 M in doubly
distilled water were prepared just before use and then diluted accordingly with water. The
supporting electrolytes were 10-3 M HCl, 0.1 M HCl, sodium phosphate buffer solution 50
mM + 0.3 M NaCl pH 8.5 and sodium acetate buffer solution 0.2 M + 20 mM NaCl pH 4.7.
Apparatus
Cyclic and square wave voltammetric measurements were performed using a PalmSens
potensiostat purchased from IVIUM Technologies (The Netherlands, www.ivium.nl) and
PalmSensPC software. The working electrodes were: a) a hanging mercury drop electrode
(EA 290 Metrohm, www.metrohm.com, type 6.0335.000, surface area 2.22 mm2) and b) a
carbon paste electrode with 3 mm inner and 9 mm outer diameter of the PTFE sleeve. The
reference electrode was Ag / AgCl / 3 M KCl and the counter electrode was a platinum wire.
Preparation of the Working Electrodes
Carbon paste electrode
The carbon paste was prepared by hand-mixing 375 mg graphite powder with 0.25 mL
mineral oil, until gaining a homogenous mixture. The resulting paste was packed tightly into a
PTFE sleeve. Electrical contact was established with a stainless steel screw. The surface was
polished to a smooth finish before each experiment. The constructed electrode was washed
with distilled water and then was transferred to supporting electrolyte solution.
Hanging mercury drop electrode
The hanging mercury drop electrode was transferred to de-aerated supporting electrolyte
solution. Initially the solution was bubbled with argon for 300 s, in order to extract oxygen
3
from it. Additionally the blank background buffer was de-aerated for 60 s before each
measurement.
Voltammetric measurements
Cyclic voltammetry (CV)
Cyclic voltammetry was performed, in different electrolytes (10-3 M HCl, 0.1 M HCl,
50 mM sodium phosphate + 0.3 M NaCl pH 8.5, 0.2 M sodium acetate buffer + 20 mM NaCl
pH 4.7) and scan rates (0.03 – 0.1 V.s-1). When using HMDE, the conditions were the
following: Ebegin = -0.2 V, Eend = -1.5 V, Estep = 0.005 V, Nscans = 3. When CPE was used, the
conditions were the following: Estart = 0 V, Eend = 1.2 V, Estep = 0.005 V, Nscans = 3.
Square wave voltammetry (SWV)
The study was performed in phosphate buffer pH 8.2, when HMDE was used. The conditions
were Estep = 0.005 V, Epulse = 0.025 V and frequency = 25Hz. The potential was scanned from
–0.1 V to –1.5 V for reduction peaks and reversibly for the anodic peaks. The potential of
deposition varied between 0 – (–1.6) V, while the deposition time varied between 0 – 300 s.
The reactions were performed in acetate buffer pH 4.7, when CPE was used. The conditions
were Estep = 0.005 V, Epulse = 0.025 V and frequency = 25Hz. The potential was scanned from
1.2 V – (–0.1) V for reduction peaks and reversibly for the anodic peaks. The potential of
deposition varied between 0 – 1.4 V, while the deposition time varied between 0 – 300 s.
Results and discussion
Cyclic voltammetry
Cyclic voltammetry was performed in a variety of supporting electrolytes (10-3 M HCl, 0.1 M
HCl, 50 mM sodium phosphate + 0.3 M NaCl pH 8.5, 0.2 M sodium acetate buffer + 20 mM
NaCl pH 4.7) in order to characterize the redox and interfacial processes of proflavine.
Cyclic polarography with HMDE
Best results were obtained by using as supporting electrolyte 50 mM sodium phosphate + 0.3
M NaCl pH 8,5 and scan rate 0.05 V.s-1. A reversible peak appears at –1.0 V and an
irreversible oxidation peak at –0.6 V (Fig. 2).
4
Cyclic voltammetry with CPE
Best results were obtained by using 0.2 M sodium acetate buffer + 20 mM NaCl pH 4.7 as
supporting electrolyte and scan rate 0.05 V.s-1. A reversible peak appears at 0.2 V and an
irreversible oxidation peak at 1.0 V (Fig. 3).
Adsorptive stripping square wave voltammetry
Adsorptive stripping square wave voltammetry was investigated in cathodic and anodic scan,
since the results obtained from cyclic voltammograms showed PF to be electroactive in both
directions.
Adsorptive stripping square wave voltammetry with HMDE
Anodic adsorptive stripping square wave voltammetry with HMDE
In order to achieve optimum results, time and potential of deposition were investigated
thoroughly. For the preconcentration step –1.2 V for 180 s were selected and concentration
curves were obtained for both peaks. In Fig. 4, it is shown that during the anodic scan, peak at
–0.6 V is not affected by the preconcentration step. On the contrary peak at –1.0 V is affected.
Linearity was observed in a range of (23.4 – 3960)  10-8 M [y = 710-5 x + 0.0068, r = 0.991;
y = I (μΑ), x = CPF (10-8 M)] and (1.17 – 234)  10-8 M [y = 0.0003 x + 0.042568, r = 0.991;
y, x as above] for the two peaks respectively. For peak at –1.0 V, RSD was 5.4 % (n = 8).
Cathodic adsorptive stripping square wave voltammetry with HMDE
The procedure followed was as the anodic scan and parameters selected in order to gain
optimum results during the preconcentration step were –1.2 V for 60 s (Fig. 5). The reduction
peak at –1.0 V was studied and no linearity was observed in the range of (1.17 – 3050)  10-8
M. In addition for concentrations above 105  10-8 M the peak splits.
Adsorptive stripping square wave voltammetry with CPE
Anodic adsorptive stripping square wave voltammetry with CPE
Time and potential of deposition were investigated as in the AdS-SWV at HMDE. Optimum
conditions for the preconcentration step were 1 V for 120 s. Using these conditions calibration
curves were obtained for both peaks. It is shown in Fig. 6 that the oxidation peak at 1 V is not
affected by the preconcentration step, whereas the peak at 0.2 V is affected. Linearity was
observed in a range of (129 – 1170)  10-8 M [y = 0.0022 x – 0.0659, r = 0.998; y, x as above]
5
and (0.2 – 23.4)  10-8 M (y = 0.0325 x + 0.0414, r = 0.996; y, x as above] for the two peaks
respectively. For peak at 0.2 V, RSD was 2.1 % (n = 8).
Cathodic adsorptive stripping square wave voltammetry with CPE
After following the same procedure as before, preconcentration at 1 V for 180 s was selected.
At these conditions, calibration curve was made for the reduction peak at 0.2 V (Fig. 7). The
peak was affected by the preconcentration as the oxidation peak at 0.2 V. Linearity was
observed in a range of (1.17 – 117)  10-8 M [y = 0.0207 x + 0.0589, r = 0.999; y, x as above].
For this peak RSD was 3.3 % (n = 8).
Conclusions
A new electroanalytical method for the determination of the intercalating agent proflavine was
developed. This electrochemical method is sensitive, fast and low cost. Both CPE and HMDE
give satisfactory results with adsorptive stripping square wave voltammetry.
References
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Captions
Fig. 1: Structure of proflavine
Fig. 2: Cyclic voltammogram of PF (CPF = 6.4*10-6 M) using HMDE, scan rate = 0.07 V/s. Other
experimental conditions as described in the experimental part 3.1.
Fig. 3: Cyclic voltammogram of PF (CPF = 1.6*10-6 M) using CPE, scan rate = 0.05 V/s. Other
experimental conditions as described in the experimental part 3.1.
Fig. 4: AdS - SW voltammograms using HMDE. Anodic scan with increasing concentration of PF (a)
supporting electrolyte, (b) CPF = 1.1710-7 M, (c) CPF = 5.910-7 M, (d) CPF = 1.6410-6 M, (e) CPF =
2.3410-6 M. Preconcentration step: -1.2 V for 180 s. Other experimental conditions as described in the
experimental part 3.2.1.
Fig. 5: AdS - SW voltammograms using HMDE. Cathodic scan with increasing concentration of PF (a)
supporting electrolyte, (b) CPF = 5.910-8 M, (c) CPF = 2.3410-7 M, (d) CPF = 5.910-7 M. Preconcentration
step: -1.2 V for 60 s. Other experimental conditions as described in the experimental part 3.2.1.
7
Fig. 6: AdS - SW voltammograms using CPE. Anodic scan with increasing concentration of PF (a)
supporting electrolyte, (b) CPF = 2.3410-8 M, (c) CPF = 1.1710-7 M, (d) CPF = 2.3410-7 M, (e) CPF =
5.910-7 M. Preconcentration step: 1 V for 120 s. Other experimental conditions as described in the
experimental part 3.2.2.
Fig. 7: AdS - SW voltammograms using CPE. Cathodic scan with increasing concentration of PF (a)
supporting electrolyte, (b) CPF = 2.3410-7 M, (c) CPF = 7.0310-7 M, (d) CPF = 1.1710-6 M, (e) CPF =
2.3410-6 M. Preconcentration step: 1 V for 180 s. Other experimental conditions as described in the
experimental part 3.2.2.
8
Figures
H2N
N
NH 2
Fig. 8
Fig. 9
9
Fig. 10
Fig. 11
10
Fig. 12
Fig. 13
11
Fig. 14
12