Damjanovi et al.
1261
Scheme 1.
bromazepam was not studied yet, the aim of this work was
to quantitatively define the multiple equilibria between all
the species actually present in the system.
Experimental
Apparatus and reagents
A GBC 911A Spectrophotometer (GBC Scientific Equip-
ment Pty Ltd.) with 1 cm silica cells was used for spectro-
photometric measurements.
A
PHM240 pH meter
(Radiometer) with a combined GK2401B electrode (Radi-
ometer) served for pH value measurements. Titrations were
performed with a TTT-60 titrator with an ABU-12 auto
burette (Radiometer). Measured pH values (pH > 2) were
converted into pcH according to relation (12): pcH
=
–log[H3O+] = pH – A. For I = 0.1 mol L–1 (NaCl) and t =
25 °C, the correction factor value was A = 0.12. For pH < 2,
pcH values were calculated according to HCl solution con-
centrations.
Bromazepam (Hoffmann La Roche) was of pharmaceuti-
cal purity grade, and other reagents, hydrochloric acid, so-
dium hydroxide, sodium chloride, sodium acetate, potassium
dihydrogenphosphate, sodium hydrogenphosphate dihydrate,
and ethanol were of analytical reagent grade (Merck). All
solutions were prepared with double distilled water.
HCl and NaOH solutions were potentiometrically stan-
dardized. NaOH solution was standardized with potassium
hydrogen phthalate. HCl solution was standardized in rela-
tion to the standard NaOH solution.
prepared in pcH range 1.2–2.0, (iii) and H2B+ solution in ac-
etate buffer (pH 4.5). Solutions in the pcH interval 1.2–2.0
and the solution of the H2B+ form were prepared with the
fast working procedure to avoid a diazepine ring-closing re-
action. The absorption spectra of all solutions were recorded
immediately after preparation in the 200–400 nm wave-
length range with the scanning speed of 1000 nm/min. For
the KaB1 determination measurements were done at 237 nm.
For the Kh1 and Kh3 determination, a stock solution
of bromazepam was prepared in ethanol (cBz = 2.64 ×
10–3 mol L–1). Aliquots of 1 mL were transferred into 50 mL
volumetric flasks and diluted to volume with HCl and NaCl
solutions to reach pH in the 1.3–3.8 range and ionic strength
I = 0.1 mol L–1. These solutions (cBz = 5.28 × 10–5 mol L–1)
were hydrolyzed at 25 °C until complete equilibration was
achieved (4 h). UV spectra were recorded in the 200–
400 nm wavelength range. Spectra of “pure” bromazepam
forms, protonated (H2A+) and molecular (HA), were re-
corded in 1 mol L–1 HCl and 0.1 mol L–1 NaCl (pH 6.8), re-
spectively. The spectrum of the H2A+ form was recorded
with a fast working procedure to minimize errors due to the
hydrolytic changes in molecular structure. The preparation
of the H3B2+ form solution and recording of its spectrum ob-
taining are described within the KaB1 determination proce-
dure. All spectra were recorded against the corresponding
blanks.
Estimation of the ring-opening reaction reversibility
A stock solution of bromazepam (Bz) was prepared in
ethanol (cBz = 1.50 × 10–2 mol L–1). One mL aliquots of
stock solution were transferred into 25 mL volumetric flasks
and diluted to volume with HCl solutions of different con-
centrations (10–3, 10–2, 0.1, and 1 mol L–1). Solutions were
thermostated (light protected) for 4 h (until the equilibrium
was established) at t = 25.0 0.1 °C (to determine the time
needed for equilibrium to establish, time-dependent spectral
changes in the absorption spectra of bromazepam were mon-
itored; depending on solution acidity, equilibrium is estab-
lished in 2–4 h). One mL aliquots of these solutions were
rapidly transferred into 25 mL of phosphate buffer (c =
0.5 mol L–1, pH 6.8, cBz = 2.40 × 10–5 mol L–1). The time-
dependent ring-closing reaction was spectrophotometrically
monitored in the 200–400 nm wavelength range, with the
appropriate blanks and the scan speed of 500 nm min–1. For
the comparison, a spectrum of bromazepam (cBz = 2.40 ×
10–5 mol L–1) in phosphate buffer (pH 6.8) was also re-
corded.
Equilibrium constants determination
Equilibrium constants KaB1, Kh1, and Kh3 (Scheme 1) were
determined spectrophotometrically at t = 25.0 0.1 °C.
KaB1 was determined according to the following procedure:
bromazepam was dissolved in 1.0 mol L–1 HCl (cBz = 2.64 ×
10–3 mol L–1) and hydrolyzed at 25 °C. After equilibration
was completed (2 h), this solution served for the preparation
of the following solutions (for all of them c = 5.28 ×
10–5 mol L–1): (i) solution of the H3B2+ form prepared in
1.0 mol L–1 HCl, (ii) mixture of the H3B2+ and H2B+ forms
+
The acidity constant of the -NH3 in the opened-ring form
(KaB2) was estimated potentiometrically with a fast working
procedure. Bromazepam was dissolved in 0.1 mol L–1 HCl
(cBz = 2.00 × 10–3 mol L–1) and hydrolyzed for 4 h at 25 °C.
To 20 mL aliquots of this solution, a standard solution of
NaOH (c = 1.15 mol L–1) was rapidly added until pH 8.5–
9.5 was reached and solutions were rapidly titrated with
0.02 mL increments of standard HCl solution (c =
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