1.

Introduction

There are generally following modes to treat wastewater i.e. physical mode, chemical
mode, biological mode and energy intensive method. Physical mode for treatment of
wastewater is that kind of technology which consist mainly separation techniques of solid-
liquid, in which filtration is the primary role for separation. Filtration method can be
classified into two parts: 1) Conventional and 2) Non-conventional method. Next mode
which is chemical mode needs several types of chemicals to get react with the pollutants of
the wastewater which have to terminate from the wastewater. Chemicals could be applied
into the wastewater for the removal, destruction or dissolution of pollutants from the
wastewater. Last mode is some kind of emerging technologies i.e. photo catalysis, by using
membrane, enzymatic treatment and advance oxidation process (AOP). In AOP, using some
type of energy like electrical energy called electrochemical method and it can be applied at
large scale for drinking water treatment (Panizza et al., 2007; Anand et al., 2015; Kumar et
al., 2015).

The degradation of wastewater can be reported by degradation of many parameters like

chemical oxygen demand (COD), biological oxygen demand (BOD), total organic carbon
(TOC), colour, concentration of particular pollutants etc. These parameters can be monitored
by their respectively instruments. Concentration of pollutants can be monitored by many
technology like spectroscopy, chromatography etc. In all these high performance liquid
chromatography (HPLC) is most significant instrument for monitoring of degradation of
pollutants from wastewater (Garg et al., 2014; Thiruvenkatachari et al., 2006).

2. Wastewater Degradation Monitoring By HPLC

Liquid chromatography is a separation technique that involves the placement

(injection) of a small volume of liquid sample into a tube packed with porous particles
(stationary phase) where individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity. The components of the sample are
separated from one another by the column packing that involves various chemical and/or
physical interactions between their molecules and the packing particles. The separated
components are collected at the exit of this column and identified by an external
measurement technique, such as a spectrophotometer that measures the intensity of the
colour, or by another device that can measure their amount. There are two main ways to
interpret a chromatogram (i.e. perform quantification):

i. Determination of the peak height of a chromatographic peak as measured from the baseline

ii. Determination of the peak area

In order to make a quantitative assessment of the compound, a sample with a known

amount of the compound of interest is injected and its peak height or peak area is measured.
After this, a calibration curve should be drawn between known concentration and peak area
or height with maximum regression coefficient. There is a linear relationship between the
height or area and the known amount of sample. This linear relationship can be used to
calculate the degraded concentration by that particular peak area or height of unknown
degraded concentration. So, HPLC can be used to monitor the degradation efficiencies for
wastewater treatment. Fig. 2 shows an example of calibration curve between known amount
and peak area or height which shows regression coefficient nearly equal to 1 (0.998) showing
best data fit into linear relationship. Fig. 3 shows result obtained from HPLC containing 4
peaks at different detection time which shows sample consist 4 types of compounds. The
components of a basic high-performance liquid chromatography [HPLC] system are shown in
the simple diagram in Fig. 4.

Figure 2. Calibration curve for HPLC (Pramod et al., 2013).

Figure 3. Results obtained from HPLC (http://shula-lc.co.il/1396-2/#Introduction).

Figure 4. High-Performance Liquid Chromatography (HPLC) system

(http://www.waters.com/waters/en_IN/How-Does-High-Performance-
LiquidChromatography-Work%3F/nav.htm?cid=10049055&locale=en_IN).
3. Case Study

Many researchers utilized HPLC or HPLC coupled with mass spectroscopy

(HPLC/MS) to monitor wastewater degradation as well as to identify intermediate products
obtained during degradation process. Mainly C18 column and acetonitrile with water as
mobile phase was used during operation of HPLC.

HPLC (Waters 1525, USA) equipped with UV detector (Waters 2487, USA) system
was used to measure the concentration of phthalic acid. HPLC was operated at ambient
temperature in isocratic mode using C18 column. The wavelength of detector was maintained
at 240 nm and the mobile phase solution used for HPLC analysis was prepared with 91%
H2O, 7% 2-propanol and 2% acetic acid with 1.2 mL/min of flow rate. Maximum removal of
 phthalic acid (PA) 68.21%, 74.36%, 82.25% was monitored by HPLC done by
electrocoagulation (EC), peroxi-coagulation (PC) and peroxi-electrocoagulation (PEC)
process respectively (Sandhwar and Prasad, 2017).

The process of phenol electrodegradation was subjected to a quantitative assessment

of the reaction products/intermediates by means of the combined HPLC/MS analysis, carried-
out for an initial concentration of phenol: 1×10 -4 M and electrolyte conductivity of 5 mScm -1,
after 2 and 6 hours of continuous electrolysis, performed at 100 and 200 mA (SS cathode and
Ti/Pt anode were used). Hence, micro-HPLC (LC 200, Eksigent, Canada) system was
combined with QTRAP 5500 mass spectrometer (AB SCIEX, Canada), equipped with an ESI
ion source, triple quadrupole and ionic trap. Reaction products were separated by means of
Halo C18 (2.7 μm, 0.5×50 mm) chromatographic column (Eksigent, Canada) at 45 oC for the
mobile phase flow of 20 μL min -1. The combined HPLC/MS analysis was conducted on four
pre-electrolyzed samples (Sample 1: I= 100 mA, t= 2 hours; Sample 2: I= 100 mA, t= 6
hours; Sample 3: I= 200 mA, t= 2 hours; Sample 4: I= 200 mA, t= 6 hours), 20 hours after
completion of the electrolysis process. Thus, electrooxidation of phenol led to the formation
of a series of intermediates, including: hydroquinone, catechol, p-benzoquinone and maleic
acid (it should be noted that oxalic acid was not instrumentally detected due to its low
molecular mass). These substances were properly identified in the respective chromatograms,
which can be seen in Fig. 5a and 5b (Piotrowska and Pierozynski, 2017).

Figure 5. HPLC chromatograms recorded for phenol-based, electrolyzed synthetic wastewater

exhibiting detected phenol electrooxidation products: A) phenol, hydroquinone, catechol and maleic
acid; B) p-benzoquinone (e Freitas et al., 2017).
 The evolution, formation and degradation of each by-product during the
photocatalysis and photolysis processes were monitored by high-performance liquid
chromatography coupled to the hybrid mass spectrometry system. The liquid-phase
chromatograph was equipped with a binary pump (Nexera LC-30AD; Shimadzu Corporation)
and an auto sampler (SIL 30AC; Shimadzu Corporation). The mass spectrometer parameters
were the same as described above. The sample volume was 8 μL. For separation, a Nucleosil ®
100-5 CN column (250 mm × 4.6 mm × 5 µm particle diameter) (Sigma Aldrich, Sao Paulo,
Brazil) was used, and as mobile phases, water (A) and acetonitrile (B), both contained 0.1%
formic acid, were used at a flow rate of 1 mLmin −1. The mobile phase was split so that the
flow became 0.2 mLmin−1 prior to entering the electrospray capillary at the mass
spectrometer, and the gradient programme started with 30% B, rising to 90% B over 16 min.
Then, the gradient was decreased to 30% B up until 22 min, which was then held for 1 min at
30% B. At the end of the chromatographic run, the column was re-equilibrated to the initial
conditions and stabilised for 4 min, which led to a total run time of 27 min. There was almost
complete removal of captopril, with 93.5% removal efficiency during photolysis and 99.9%
during photocatalysis (e Freitas et al., 2017).

Reference:

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Anand M.V., Srivastava V.C., Singh S., Bhatnagar R., Mall I.D., Electrochemical treatment
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Kumar S., Singh S., Srivastava V.C., Electro-oxidation of nitrophenol by ruthenium oxide
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Garg K.K., Prasad B., Srivastava V.C., Comparative study of industrial and laboratory
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Thiruvenkatachari R., Ouk Kwon T., Shik Moon I., Degradation of phthalic acids and
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Piotrowska G., Pierozynski B., Electrodegradation of Phenol through Continuous Electrolysis

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e Freitas J.R., Quintao F.J., Silva J.C., Silva S.D., Aquino S.F., Afonso R.J., Characterisation
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