4
86
S. Chakma, V.S. Moholkar / Ultrasonics Sonochemistry 29 (2016) 485–494
Sigma–Aldrich India Ltd. Mono-potassium phosphate (KH
di-potassium phosphate (K HPO and hydrogen peroxide
, 30% v/v) were procured from Merck India Ltd. All solutions
were prepared in ultrapure Milli-Q water (resistivity P 18 MO cm
at 298 K).
2
PO
4
),
ultrasound waves in the bath and the reaction mixture inside the
reaction tube was found to be same, which indicated negligible
attenuation of ultrasound waves through the wall of reaction test
tube. Due viscous dissipation of energy of ultrasound waves during
propagation, the temperature of the water in the bath rises. This
rise was controlled by circulation of water from a temperature con-
trolled cooling water circulating bath (Amkette Analytics, Model:
WB2000). The temperature of the reaction mixture in the test tube
was same as that of the water in the bath.
2
4
)
2 2
(H O
2.2. Activity and Bradford assay for HRP enzyme
Activity of HRP enzyme was determined as per standard proto-
col (Sigma–Aldrich, EC 1.11.1.7) [26] with pyrogallol as substrate.
The following reaction determines the activity of HRP enzyme:
The IBP degradation experiments were conducted in four cate-
gories, viz. (1) enzymatic treatment with mechanical stirring, (2)
sonolysis, (3) sono-enzymatic treatment at atmospheric static
pressure, and (4) sono-enzymatic treatment at elevated static pres-
sure of 200 kPa. In categories 2, 3 and 4 involving sonolysis, exper-
iments were conducted at two frequencies (either 37 or 80 kHz). In
category 4 experiments, the test tube containing reaction mixture
was connected to a nitrogen cylinder through a side nozzle. The
mouth of the test tube was sealed with threaded cap (please refer
to the schematic of experimental set-up provided in Supplemen-
tary material). During withdrawal of aliquots of reaction mixture,
the pressure in reaction test tube was released. The static pressure
was restored to original level, as the sonication was resumed. The
rationale underlying the technique of elevating static pressure of
reaction mixture will be explained subsequently. The actual pres-
sure amplitude of ultrasound waves reaching the reaction mixture
is a strong function of wall thickness of the test tube holding reac-
tion mixture. In order to keep this important parameter constant,
same test tube has been used in all experiments.
HRP
Pyrogallol ðdonorÞþH
2
O
2
ꢀ! 2H OþPurpurogallin ðoxidized donorÞ
2
The assay composition was as follows: ultrapure water 2.1 mL,
phosphate buffer (pH 7, 100 mM) 0.32 mL, hydrogen peroxide
solution (diluted to 60:1) 0.16 mL, pyrogallol solution 0.32 mL,
enzyme (HRP) 0.1 mL. After mixing of above solutions, the final
concentrations in 3 mL mixture were: 14 mM of phosphate buffer,
0
2 2
.027% (w/w) H O , 0.5% (w/v) pyrogallol. These concentrations
were maintained same in all experiments of IBP degradation. The
absorbance of purpurogallin was recorded every 20 s at 420 nm
using UV–Vis spectrophotometer (ThermoFischer, Model:
UV-2300). The activity of the enzyme was calculated using the
following formula:
ð
D
A
420=20sÞ ꢁ V
t
ꢁ D
f
Unit=mL Enzyme ¼
Notation:
e
ꢁ
m
S
V
t
–
total volume of reaction mixture (3 mL),
e – extinction coefficient (12 for purpurogallin
The total treatment time was 60 min and the total reaction
volume in a typical experiment was 15 mL. In this volume, concen-
trations of different components were as follows: IBP = 10 ppm (or
D
f
– dilution factor,
at 420 nm), – volume of sample (0.1 mL).
v
s
ꢀ
2
The protein content of the HRP enzyme was also determined by
Bradford assay. Bovine serum albumin (BSA) was used as the stan-
dard protein. 3 ml of Bradford reagent and 0.1 ml HRP enzyme
were mixed to form Bradford assay mixture. The mixture was kept
in incubation for 15 min and the absorbance in mixture was mea-
sured at 595 nm. With this procedure, the protein content of HRP
was determined as 0.0228 mg/mL.
4.76 ꢁ 10 mM), H O = 8.55 mM, HRP = 0.08 U/mL, phosphate
2
2
buffer (pH 7) = 14 mM. The pH of the reaction mixture was
maintained at 7 in all experiments. This pH was optimum for the
highest activity of enzyme (for results of optimization, refer to
the Supplementary material, Fig. S2). To monitor the time profile
of IBP degradation, aliquots of reaction mixture (200 lL) were
withdrawn at regular intervals and were analyzed for the residual
IBP. After completion of the treatment, aliquotes of reaction
mixture were analyzed for identification of intermediates of IBP
degradation.
2.3. Experimental setup and procedure
Purely enzymatic reactions were carried out under well stirred
conditions using mechanical stirring with a small magnetic bar
length = 1 cm, diameter = 0.5 cm) at 150 rpm. A schematic of the
2.4. Analytical methods
(
experimental setup is given in Supplementary material (Fig. S1).
Experiments were carried out in a custom built (20 mL) test tube
made of borosilicate glass (O.D. = 16 mm, length = 150 mm). The
experiments with sonication were conducted in an ultrasound bath
The concentration of IBP in the aliquot of the reaction mixture
was analyzed using High Performance Liquid Chromatography
Shimadzu, Model: SPD-20A) equipped with C-18 reverse phase
m) and UV detector at 220 nm.
(
column (250 mm ꢁ 4.6 mm, 5
l
(
Elmasonic, Germany, Model: P-30H). The ultrasound bath could
operate at two frequencies, viz. 37 kHz (130 W) and 80 kHz
100 W). Approximately 2/3rd volume of the bath was filled with
The mobile phase was a mixture of acetonitrile and aqueous acetic
acid 0.1% (60/40, v/v) with an isocratic flow of 1.5 mL/min. In order
to determine the mineralization, the total organic carbon (TOC) in
the IBP reaction solution was measured with Aurora TOC analyzer
(
water that acted as medium for transmission of ultrasound waves.
The test tube with reaction mixture was placed exactly at the cen-
ter of the ultrasound bath and it was immersed to approx. 75% of
its height. This ensured that entire reaction mixture in the test tube
was exposed to ultrasound waves. The position of test tube in the
bath was maintained carefully same in all experiments to avoid the
artifacts in experimental results due to spatial variation of acoustic
intensity [27,28]. The actual power input to the ultrasound bath
and to the reaction mixture inside the test tube was determined
using calorimetric technique [29,30]. On this basis, the exact volu-
metric energy dissipation for 37 and 80 kHz were as follows:
(
O-I-Analytical, Model: 1030). The intermediates formed during
sono-enzymatic degradation of IBP were identified using GC–MS
Varian 240-GC) after liquid–liquid extraction with dichloro-
methane. 1 L of extracted sample was injected with a split ratio
(
l
of 10:1. Helium gas was used as a carrier and the mass detector
worked in scan mode with m/z range of 50–600. The GC–MS
spectrums are given in Supplementary materials (Figs. S5–S9).
2.5. Experimental data analysis
0
.0174 W/mL (for 37 kHz) and 0.0116 W/mL (for 80 kHz). The cor-
responding pressure amplitudes of ultrasound waves in the bath
for frequencies of 37 and 80 kHz were calculated as 200 kPa and
The kinetic constants of IBP degradation were determined by fit-
ting pseudo 1st order model to the kinetic data of IBP degradation.
Using these kinetic constants, activation energy for IBP degradation
1
70 kPa, respectively. Moreover, the pressure amplitudes of