Selected Drugs by Solid-Phase Extractionprr.hec.gov.pk/jspui/bitstream/123456789/8196/1/Tahira...
145
Hospital Wastewater Screening for Pharmaceutical Compounds and Removal of Selected Drugs by Solid-Phase Extraction THESIS SUBMITTED TOWARDS THE PARTIAL FULFILMENT OF THE REQUIREMENT OF THE UNIVERSITY OF SINDH, FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE IN ANALYTICAL CHEMISTRY TAHIRA QURESHI National Centre of Excellence in Analytical Chemistry, P T
Transcript of Selected Drugs by Solid-Phase Extractionprr.hec.gov.pk/jspui/bitstream/123456789/8196/1/Tahira...
Hospital Wastewater Screening for
Pharmaceutical Compounds and Removal of
Selected Drugs by Solid-Phase Extraction
THESIS SUBMITTED TOWARDS THE PARTIAL FULFILMENT OF
THE REQUIREMENT OF THE UNIVERSITY OF SINDH, FOR THE
AWARD OF
DOCTOR OF PHILOSOPHY DEGREE IN ANALYTICAL CHEMISTRY
TAHIRA QURESHI
National Centre of Excellence in Analytical Chemistry,
P T
University of Sindh, Jamshoro Pakistan
2017
3
CERTIFICATE
This is to certify that the work present in this thesis entitled “Hospital wastewater screening for
pharmaceutical compounds and removal of selected drugs by solid-phase extraction” has been
carried out by Tahira Qureshi under our supervision. The work is genuine, original and, in our
opinion, suitable for submission to the University of Sindh for the award of degree of PhD in
Analytical Chemistry.
SUPERVISOR
___________________________________________
Dr. Najma Memon
Professor
Supervisor
National Centre of Excellence in Analytical Chemistry, University of
Sindh, Jamshoro, Pakistan.
CO-SUPERVISOR
___________________________________________
Dr. Saima Q. Memon
Associate Professor
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Co-Supervisor
Dr.M.A.Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan.
DEDICATION
It is with deepest love, respect, and admiration that I dedicate this
dissertation to my parents whose support and constant love have sustained
me throughout my life and to my brilliant, always encouraging and ever
supportive teachers.
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ACKNOWLEDGEMENTS
I am grateful to Almighty Allah (S.W.T.), the supreme, the merciful, the most gracious,
the compassionate, the beneficent, Who is the entire and only source of every knowledge and
wisdom gifted to mankind and Who blessed me with the ability to do this work.
I would wholeheartedly thank to my supervisors, Prof. Dr. Najma Memon and Dr.
Saima Q. Memon for their guidance and support throughout this study, and especially for their
confidence in me. I could not achieve this goal without their thought provoking guidance,
cooperation and moral support. They have always been a source of inspiration and a role model
for me. Their patience and generosity has enabled me to overcome all the hurdles coming in
the way of success. Their teachings have not only improved my research skills but also refined
me as human.
I would like to thank my teacher Dr. Amber R. Solangi, whose compassionate view of
the world gave me a deeper perspective of what is truly important.
I am thankful to Director, National Centre of Excellence in Analytical Chemistry, Prof.
Dr. Shahabuddin Memon for boosting my moral and providing good research facilities.
I am also gratified to Prof. Tasneem Gul Kazi, Prof. Syed Tufail Hussain Sherazi, Prof.
Sirrajuddin, and all faculty members for their research consultancy and cooperation. I
acknowledge all the administrative and non-teaching staff for their cooperation and support.
I would like to thank my dearest friend Dr. Huma Ishaque for your understanding and
encouragement in every moments of need. Your friendship makes my life a wonderful
experience. I am grateful to my group fellows Rabel Soomro, Sidra Amin, Ruqaya Shaikh,
Sanam Soomro, Fouzia Chang, Paras Azadi and Zakir Hussain at NCEAC, for their teamwork
and support. Also, I would like to thank Dr. Kamran Abro, I express my heartfelt gratefulness
for his assistance and support at PCSIR, Lab. Karachi, Pakistan.
I would like to acknowledge the Higher Education Commission of
Islamabad, Pakistan for providing funds through ‘Indigenous Fellowship Program batch VII’
[117-8945-PS7-141]
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At last but not the least, I acknowledge and offer my heartiest gratitude to my beloved
parents, brothers and sisters for their moral support, cooperation, encouragement, patience,
tolerance and prayers for my health and success during this work.
Tahira Qureshi
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ABSTRACT
Hospital waste poses a significant impact on health and environment. Many pharmaceuticals,
some with endocrine disrupting functions, enter sewage systems and can pass through the
sewage treatment system to be discharged to the wider environment if not fully removed in the
treatment process. Sources include domestic residences and hospitals, the latter being a
potential point source of contaminants. Generally, hospital wastewater is differentiated as
hospital sewage, infective and potentially infective wastewater, chemical wastewater, and
radioactive wastewater. Pharmaceuticals residue in sewage and in the aqueous environment has
begun to attract attention several scientists world over. There are several reports on the
persistence of pharmacceutially active compounds (PhACs) in water streams around the globe.
However, no such study is conducted in Pakistan; therefore this thesis was aimed to investigate
persistence of PhACs and possible removal solutions. Study area for persistent PhACs was
hospital in city of Hyderabad, Pakistan. Following were the aims and objectives of current
research
1. Surveying local hospitals to identify frequently prescribed pharmaceuticals.
2. Monitoring hospital wastewater for screening of PhACs.
3. Selection of PhACs on the basis of above study and studies reported in literature.
4. Performance of adsorption experiments to estimate adsorption capacity, kinetics of
selected natural adsorbents for selected PhACs.
5. Utilization of modified natural adsorbents for removal of selected PhACs.
To address the objectives set above, local hospitals in Hyderabad, Sindh and its vicinity were
surveyed to collect the information about frequently prescribed drugs. On the basis of surveyed
data and reported literature, highly persistent PhACs were selected. From surveyed and
reported literature, five antibiotics (cefuroxime, cefotaxime, cefradine, ofloxacin and
ciprofloxacin) and two analgesics (ibuprofen and diclofenac sodium) were shortlisted for
further studies.
Initial screening of ofloxacin, ciprofloxacin and diclofenac from hospital wastewater was
performed by LC-MS methods. Concentration of these drugs was found within the following
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ranges: OFL 17-53 µg L-1, CIP 70-164 µg L-1 and DFS 78 - 118 µg L-1. IBP was determined
to be in the range of 0.052-0.080 µg L-1. The presence of OFL, CIP and DFS were found in µg
L-1level in nearly all of the samples.
Cephalosporins type antibiotics are widely used to treat infectious diseases. A new high
performance liquid chromatographic method for the determination of three cephalosporins
(cefradine, cefuroxime and cefotaxime) was developed. Under optimised conditions, all three
cephalosporins were baseline separated within 5 min. Linear responses for cefradine 5-20 g
mL-1, cefuroxime 0.5-15 g mL-1 and cefotaxime 1.0-20 g mL-1 were established. LOD of
0.05-0.25 µg mL-1 after preconcentration was achieved. The method was applied to screen the
selected cephalosporins from hospital wastewater samples. Moreover, method was applied to
study stability of aqueous solutions and acid/base induced degradation of all three drugs.
Ibuprofen (IBP) is one of the most used active pharmaceutical ingredients globally. Due to its
extensive use and resistance to biodegradation it is considered as environmental pollutant and
included in the list of pharmaceutically active compounds (PhACs). A GC-MS method was
developed along with Solid-phase extraction (SPE) for clean-up and enrichment of samples.
The instrumental calibration range for IBP was found 0.8 to 70 µg mL-1. After preconcentration
LOD of 0.8 ng mL-1 and LOQ of 2.6 ng mL-1 was achieved. The method was applied to
determine IBP in synthetic, hospital and municipal wastewaters and river water. It may be
concluded that GC/MS is useful tool for quick identification and determination of IBP in
aqueous environmental samples.
Screening data reveals persistence of DFS, IBP, CIP and OFL in hospital wastewater of
Hyderabad. Therefore sorptive removal methods using biobased sorbents were tested for their
sorption activity. Sawdust, peanut shell and hydrothermal carbonized Ziziphus mauritiana L.
fruit were used in this study to prioritize sorbent. Processes of sorbents are surmised below.
Sawdust is waste generated in woodworks, due to its fine particle size and easy availability
makes it attractive to be used as sorbent. Sawdust water, acid and base washed was tested for
its sorption efficiency towards ofloxacin. HCl treated sawdust was found to have maximum
removal efficiency (96%) with the sorption capacity of 47 µmol g-1 as compared to other treated
sorbents. Amount of sorbent have significantly positive impact on the removal for all three
treated sorbents whereas concentration of sorbate has non-significant positive effect for HCL
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treated sawdust. Further, sorption isotherms, kinetics and thermodynamics studies onto HCL
treated sawdust showed that reaction is exothermic and spontaneous in nature and psuedo-
second order is predominant route. Complex sorption mechanism with simultaneous intra -
particle diffusion as well as surface adsorption phenomena is responsible for sorption of
ofloxacin onto sawdust.
Peanuts shells (PS) were treated with acid and alkali wash for CIP sorption. Langmuir isotherm
model was dominant for acid treated PS, however for alkali treated PS Freundlich isotherm
model is well fitted but the obtained mean sorption energy (E) by D-R isotherm, is under the
magnitude of the physisorption process. Possibly, the treated PS showed heterogenous sorbent
surface behavior trend due to different interaction strengths and adsorption energy of CIP onto
treated PS. The maximum sorption capacities were observed by Langmuir isotherm were 42.2
µmol g-1 and 10.12 µmol g-1 onto acid treated PS and alkali treated PS, respectively. The acid
treated PS shows better adsorption capacity than the alkali treated PS. CIP adsorption shows a
maximum at pH 8 because of the electrostatic interaction between positively charge CIP and
negatively charged acid treated PS.
Hydrothermal carbons are new generation of sorbent materials obtained through carbonization
of cellulosic or lignocellulosic biomass under hydrothermal conditions and endogenous
pressures. Wild variety of Ber fruit abundantly available in Sindh region of Pakistan was used
as source material to prepare
HTC. The IBP and DFS simultaneous removal were done onto HTC-ZM. Set of 18 experiments
was used and factors as pH, amount of sorbent, contact time and concentration of sorbate were
considered the critical factors to be studied for removal. Maximum sorption occurred at pH
4.0 for both PhACs. From linear plot of D-R isotherm, capacity was found 2030 µmol g-1 for
DFS and 2540 µmol g-1 for IBP onto HTC-ZM. The mean free energy for both, DFS and IBP
was 8.1 KJ mol-1 and 8.3 KJ mol-1 respectively, specifying a physiosorption process. Kinetics
equations predicted a complex nature yet efficient sorption process. It may be concluded that
natural sorbents like sawdust and peanut shell with acid treatment could be employed as sorbent
for removal of selected quinolones as compared to alkali treatment of sorbents. HTC-ZM was
found to be good sorbent for removal of PhACs.
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TABLE OF CONTENTS
I
DEDICATION II ACKNOWLEDGEMENTS
III
ABSTRACT V
TABLE OF CONTENTS IX
LIST OF TABLES XVIII
LIST OF FIGURES XXI
ABBREVIATIONS XXIV
CHAPTER 1 INTRODUCTION 1
1.1 EMERGING POLLUTANTS IN WATER SYSTEMS 1
1.2 PHARMACEUTICALLY ACTIVE COMPOUNDS IN
ENVIRONMENTAL WATER SYSTEMS 2
1.3 PHARMACEUTICALLY ACTIVE COMPOUNDS AS CONTAMINANT 2
1.4 PERSISTENT PHARMACEUTICALLY ACTIVE COMPOUNDS IN WATER
SYSTEMS 3
1.5 CLASSIFICATION OF DRUGS 4
1.5.1 ANTIBIOTICS 4
1.5.2 ANALGESICS 5
1.6 SOURCES OF PHARMACEUTICALLY ACTIVE COMPOUNDS IN WATER
SYSTEMS 5
CERTI
12
1.7 IMPACT OF PHARMACEUTICALLY ACTIVE COMPOUNDS ON HUMAN
HEALTH AND AQUATIC ENVIRONMENT 8
1.8 REMOVAL TECHNOLOGIES FOR PERSISTENT PHARMACEUTICALLY
ACTIVE COMPOUNDS IN WATER SYSTEMS 9
1.9 SORPTION STUDIES 11
1.9.1 LANGMUIR ADSORPTION ISOTHERM 12
1.10
MATHEMATICAL AND
STATISTICAL APPROACH FOR REMOVAL TECHNOLOGIES 13
1.10.1.1 CENTRAL COMPOSITE DESIGNS (CCD) 14
1.12
DETERMINATION TECHNIQUES FOR PHARMACEUTICALLY ACTIVE
COMPOUNDS IN WATER SYSTEM 15
1.13 INSTRUMENTATION 16
1.13.1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
1.9.2 FREUNDLICH
ADSORPTION ISOTHERM
12
1.9.3 DUBININ–RADUSHKEVICH
ADSORPTION
ISOTHERM 13
1.11 CURRENT SITUATION OF
PHACS IN PAKISTAN
14
1.13.2 LIQUID CHROMATOGRAPHY
MASS
SPECTROMETRY (LC/MS) 17
1.13.3 GAS
CHROMATOGRAPHY MASS
SPECTROMETRY
(GC/MS) 18
1.13.4 SOLID PHASE
EXTRACTION (SPE) 19
1.13.5 ULTRAVIOLET/VISIBLE
SPECTROSCOPY
20
1.13.6 SCANNING ELECTRON
MICROSCOPE (SEM) 21
13
( HPLC) 16
1.13.7 BRUNAUER, EMMETT AND TELLER (BET)
ANALYZER 23
1.14. AIMS AND OBJECTIVES OF CURRENT RESEARCH STUDY 24 1.14.1
OUTPUT OF RESEARCH STUDY 25
1.15 STRUCTURE OF THESIS 25
CHAPTER 2 LITERATURE REVIEW
26
2.1 WORLDWIDE SITUATION OF PHACS IN
AQUATIC ENVIRONMENT 26
2.2 SCENARIO OF PAKISTAN’S WATER SYSTEM
ENCOMPASSING PHACS 28
2.3 EFFECTS OF PHACS ON LIVING BEING 29
2.4 ANALYTICAL METHODOLOGY OF SELECTED PHARMACEUTICAL
COMPOUNDS 30
2.4.1 ANTIBIOTICS 31
2.4.2 ANALGESICS AND ANTI-INFLAMMATORY DRUGS 32
2.5 REMOVAL OF PHARMACEUTICALS FROM WATER SYSTEMS 34
2.6 SUMMING UP LITERATURE REVIEW 37
CHPATER 3 RESEARCH METHODOLOGY 38
3.1 RESEARCH WORK PLAN 38
3.2 CHEMICALS AND SOLVENTS 40
14
3.3 INSTRUMENTATION 41
3.4 LC/MS SCREENING OF OFLOXACIN, CIPROFLOXACIN AND DICLOFENAC SODIUM
FROM HOSPITAL WASTEWATER IN HYDERABAD SINDH (PAKISTAN) 43
3.4.1 SAMPLING 43
3.4.2 COLLECTION OF SAMPLES 43
3.4.3 SAMPLE PRE-TREATMENT OF HOSPITAL
WASTEWATER 44
3.4.4 SOLID-PHASE EXTRACTION 44
3.4.5 INSTRUMENTATION 44
3.5 LC/UV AND LC/MS DETERMINATION OF CEFRADINE, CEFUROXIME, AND
CEFOTAXIME 45
3.5.1 SAMPLE COLLECTION AND PREPARATION 45
3.5.2 SOLID PHASE EXTRACTION 45
3.5.3 INSTRUMENTATION 45
3.5.4 LC AND LC/MS CONDITIONS 45
3.5.5 SYNTHETIC AND HOSPITAL WASTEWATER
SAMPLES 46
3.6 DETERMINATION OF IBUPROFEN DRUG IN AQUEOUS ENVIRONMENTAL SAMPLES
BY GAS CHROMATOGRAPHY– MASS SPECTROMETRY WITHOUT DERIVATISATION
47
3.6.1 PREPARATION OF SOLUTIONS AND SAMPLES 47
3.6.2 SOLID-PHASE EXTRACTION 47
3.6.3 STANDARD SOLUTION 47
15
3.6.4 SYNTHETIC WASTEWATER 48
3.6.5 CHROMATOGRAPHIC CONDITIONS FOR
DETERMINATION OF IBUPROFEN 48
3.7 REMOVAL OF OFLOXACIN ONTO SAWDUST
(ERYTHROPHLEUM SUAVEOLENS L.) 49
3.7.1 PREPARATION OF SORBENT 49
3.7.2 SAMPLE COLLECTION 49
3.7.3 SYNTHETIC WASTEWATER 49
3.7.4 SOLID-PHASE EXTRACTION 49
3.7.5 MATHEMATICAL AND STATISTICAL PROCEDURE 50
3.7.6 SORPTION EQUILIBRIUM OF OFLOXACIN ONTO
SAWDUST (SD) 50
3.8
REMOVAL OF CIPROFLOXACIN (CIP) ONTO PEANUT
SHELLS (ARACHIS HYPOGAEA L.) 53
3.8.1 PREPARATION OF SORBENT 53
3.8.2 BATCH STUDY FOR REMOVAL BY RESPONSE
SURFACE METHODOLOGY 53
3.8.3 ADSORPTION ISOTHERMS OF CIP ONTO TREATED
3.7.6.1 LANGMUIR ISOTHERM 50
3.7.6.2 FREUNDLICH
ISOTHERM 50
3.7.6.3 DUBININ-RADUSHKEVICH
ISOTHERM 51
3.7.7. ADSORPTION KINETICS
OF OFLOXACIN ONTO SD 52
16
PS 53
3.8.4 ADSORPTION KINETIC STUDIES OF CIP ONTO
TREATED PS 54
3.8.5 SURFACE CHARACTERIZATION 54
3.8.6 REMOVAL FROM SYNTHETIC WASTEWATER AND
HOSPITAL WASTEWATER 54
3.9 SIMULTANEOUS REMOVAL OF IBUPROFEN AND
DICLOFENAC SODIUM ONTO HTC OF BER (ZIZIPHUS
MAURITIANA L.) 56
3.9.1 PREPARATION OF HYDRTHERMAL CARBONEOUS
FROM BER FRUIT 56
3.9.2 SURFACE CHARACTERIZATION 56
3.9.3 BATCH ADSORPTION EXPERIMENTS 57
3.9.4 ADSORPTION ISOTHERM STUDY OF DFS AND IBP 57
3.9.5 ADSORPTION KINETICS OF DFS AND IBP 57
3.9.6 REMOVAL OF DFS AND IBP FROM SPIKED
SYNTHETIC WASTEWATER 57
CHAPTER 4 RESULTS AND DISCUSSION 59
4.1 LC/MS SCREENING OF PHARMACEUTICAL COMPOUNDS FROM
HOSPITAL WASTEWATER IN HYDERABAD, SINDH (PAKISTAN)
.......................................................................................... 60
17
4.2 DETERMINATION OF IBUPROFEN DRUG IN AQUEOUS ENVIRONMENTAL SAMPLES
BY GAS CHROMATOGRAPHY– MASS SPECTROMETRY WITHOUT DERIVATISATION
61
4.2.1 METHOD OPTIMIZATION ............................................... 62
4.2.1.1 INJECTION PARAMETERS ........................................... 62
4.2.1.2 SAMPLE VOLUME AND INJECTION MODE ................ 63
4.2.1.3 TEMPERATURE PROGRAMMING 64
4.2.1.4 DETECTOR PARAMETERS 64
4.2.2 METHOD VALIDATION ............................................... 64
4.2.3 RECOVERY OF IBP .......................................................... 65
4.2.4 REAL WATER SAMPLES ................................................. 65
4.3 LC/UV AND LC/MS DETERMINATION OF CEFRADINE, CEFUROXIME, AND
CEFOTAXIME HPLC METHOD DEVELOPMENT
................................................................................... 67
4.3.1 ANALYTICAL FIGURES OF MERIT ................................ 68
4.3.2 INDUCED HYDROLYSIS OF CEPHALOSPORINS ........... 69
4.3.3 ROOM TEMPERATURE DEGRADATION OF
AQUEOUS SOLUTIONS ............................................................ 71
4.3.4 SAMPLE PRECONCENTRATION AND CLEAN-UP ......... 71
4.3.5 SYNTHETIC AND HOSPITAL WASTEWATER
SAMPLES .................................................................................. 75
4.4 OFLOXAIC REMOVAL OPTIMIZATION ONTO TREATED
SAWDUST (ERYTHROPHLEUM SUAVEOLENS L.) BY RESPONSE
18
SURFACE METHODOLOGY (RSM) ..................................................... 77
4.4.1 OPTIMIZATION OF OFLOXACIN REMOVAL BY
SAWDUST ................................................................................. 77
4.4.2 STATISTICAL ANALYSIS AND MODEL VALIDATION . 78
4.4.3 ADSORPTION EQUILIBRIUM STUDIES.......................... 84
4.4.4 KINETICS OF ADSORPTION............................................ 86
4.4.5 THERMODYNAMICS ....................................................... 87
4.4.6 APPLICATION OF REMOVAL PROCESS ........................ 88
4.5 REMOVAL OF CIPROFLOXACIN ONTO TREATED PEANUT SHELLS (ARACHIS
HYPOGAEA L.) BY RESPONSE SURFACE METHODOLOGY (RSM)
...................................................................... 90
4.5.1 MATHEMATICAL AND STATISTICAL PROCEDURE..... 90
4.5.2 EXPERIMENTAL DESIGN ................................................ 91
4.5.3 STATISTICAL ANALYSIS FOR CIP REMOVAL .............. 91
4.5.4 ADSORPTION KINETICS AND ISOTHERM..................... 96
4.5.5 SURFACE CHARACTERIZATION .................................... 98
4.5.6 APPLICATION OF REMOVAL OF CIP FROM
HOSPITAL WASTEWATER ONTO ACID TREATED PS .......... 99
4.6 SIMULTANEOUS REMOVAL OF IBUPROFEN AND DICLOFENAC SODIUM ONTO HTC
DERIVED FROM BER (ZIZIPHUS MAURITIANA L.) FRUIT BY RSM .....................................
101
4.6.1 SURFACE CHARACTERIZATION ............................... 101
4.6.2 ADSORPTION STUDIES ................................................. 104
19
4.6.3 OPTIMIZATION OF IBP & DFS SIMULTANEOUS
REMOVAL BY FACTORIAL DESIGN ..................................... 104
4.6.4 SORPTION ISOTHERMS ................................................ 109
4.6.5 KINETICS STUDY .......................................................... 110
4.6.6 REMOVAL FROM SYNTHETIC WASTEWATER 112
CHAPTER 5 CONCLUSIONS AND
RECOMMENDATIONS
113
5.1 CONCLUSION 113
5.2 RECOMMENDATIONS 114
REFERENCES 115
PUBLISHED PAPERS 137
LIST OF TABLES
TABLE 1.1 ORIGINS AND FATE OF PPCPS IN THE ENVIRONMENT
DESCRIPTION OF LEGENDS GIVEN IN FIGURE 1.1 6
TABLE 1.2 ADVERSE EFFECTS OF PHACS 8
TABLE 2.1 SELECTED PHARMACEUTICAL COMPOUNDS AND THEIR
STRUCTURE 31
TABLE 2.2 SELECTED PHARMACEUTICAL COMPOUNDS DETECTION
IN HOSPITAL WASTEWATER REPORTED IN YEAR 2011-2015 34
20
TABLE 3.1 SURVEY FORM OF HEALTHCARE FACILITIES FOR
PHARMACEUTICALS UTILIZATION 40
TABLE 3.2 LIST OF INSTRUMENTS AND OPERATIONAL EQUIPMENTS 42
TABLE 3.3 COMPOSITION OF SYNTHETIC WASTEWATER USED FOR
RECOVERY STUDIES OF VARIOUS DRUGS 47
TABLE 3.4 LANGMUIR, FREUNDLICH AND D-R ISOTHERM
EQUATIONS AND PARAMETERS 52
TABLE 4.1 HOSPITAL WASTEWATER SCREENED SAMPLES BY LC/MSMS FOR
CIPROFLOXACIN, OFLOXACIN AND DICLOFENAC
SODIUM 60
62
TABLE 4.3 ACCURACY AND PRECISION DATA ASSAY OF IBP IN
SYNTHETIC WASTEWATER 66
TABLE 4.4 DETERMINATION OF IBP IN REAL WATER SAMPLES
FOLLOWED BY SPE 67
TABLE 4.5 INTRA-DAY AND INTER-DAY PRECISIONS FOR CEFRADINE,
CEFUROXIME AND CEFOTAXIME 70
TABLE 4.6 (A) RECOVERIES OF CEFUROXIME, CEFATOXIME AND CEFRADINE USING
SPE AT VARIOUS CONCENTRATIONS AND WITH
DIFFERENT ELUTION SOLVENTS 74
TABLE 4.6 (B) STABILITY OF DRUGS AT VARIOUS TEMPERATURES 75
TABLE4.6 (C). SAMPLE PRECONCENTRATION USING
EVAPORATION 76
TABLE 4.7 VARIABLES CODE LEVELS OF OFL REMOVAL BY CCD 78
TABLE 4.8 EXPERIMENTAL OBSERVED % SORPTION AND PREDICTED
% SORPTION OF OFLOXACIN 80
TABLE 4.9 (B) OPTIMUM PREDICTED PARAMETER VALUES AND MODEL VALIDATION 85
21
TABLE 4.10 (A) LANGMUIR, FREUNDLICH AND D-R SORPTION
ISOTHERM PARAMETERS FOR REMOVAL OF OFLOXACIN ONTO HCL
TREATED SAWDUST 86
TABLE 4.10 (B) KINETIC PARAMETERS FOR REMOVAL OF
OFLOXACIN ONTO HCL TREATED SAWDUST 88
TABLE 4.11 REMOVAL OF OFLX IN HOSPITAL WASTEWATER WATER
SAMPLES FOLLOWED BY SPE 90
TABLE 4.12 (A) VARIABLES CODE LEVELS OF DESIGN USED IN
EXPERIMENTAL FOR REMOVAL OF CIP ON TREATED PS 91
TABLE 4.12 (B) EXPERIMENTAL %SORPTION AND PREDICTED
%SORPTION OF CIPROFLOXACIN 93
TABLE 4.11 (C) OPTIMUM PREDICTED PARAMETER VALUES AND MODEL VALIDATION 97
TABLE 4.13 (A) FITTING PARAMETERS FOR LANGMUIR, FREUNDLICH AND D-R
ISOTHERM MODELS FOR ADSORPTION OF CIPROFLOXACIN
ONTO TREATED PS 98
TABLE 4.14 (B) PSEUDO-FIRST ORDER KINETIC MODEL, PSEUDOSECOND ORDER
MODEL, AND MORRIS-WEBER KINETIC MODEL
PARAMETERS FOR ADSORPTION OF SORPTION OF CIP ONTO
TREATED PS 98
TABLE 4.15 SPIKED HOSPITAL WASTEWATER SAMPLES REMOVAL
BY ACID TREATED PS 100
TABLE 4.16 (A) LEVELS OF FACTORS USED IN EXPERIMENTAL
DESIGN FOR REMOVAL OF DFS & IBP ONTO HTC-ZM 105
TABLE 4.16 (B) EXPERIMENTAL %SORPTION AND PREDICTED
%SORPTION OF DICLOFENAC SODIUM (DFS) AND IBUPROFEN (IBP)
ONTO HTC-ZM 106
22
TABLE 4.17 DESIGN VALIDATION FOR DFS & IBP REMOVAL BY
COMPARING DESIGN PREDICTED AND OBTAINED EXPERIMENTAL
RESPONSE ONTO HTC-ZM 110
TABLE 4.18 (A) FITTING PARAMETERS OF LANGMUIR ISOTHERM,
FREUNDLICH ISOTHERM AND D-R ISOTHERM FOR SORPTION
EQUILIBRIUM OF DICLOFENAC SODIUM AND IBUPROFEN 111
TABLE 4.18 (B) KINETIC PARAMETERS FOR REMOVAL OF DFS AND
IBP ONTO HTC AT CONCENTRATION LEVEL OF 3.14 Х 10-5 M, (4.85 Х
10-5 M), RESPECTIVELY. CONSTANT SHOWN IN PARENTHESIS
INDICATES HIGHER CONCENTRATIONS OF DRUGS; 3.14 Х 10-4 M AND
4.85 Х 10-4 M FOR DFS AND IBP RESPECTIVELY 112
TABLE 4.19 SYNTHETIC WASTEWATER REMOVAL OF SPIKED IBP
AND DFS ONTO HTC-ZM 113
23
LIST OF FIGURES
FIGURE 1.6 BLOCK DIAGRAM OF UV/VISIBLE SPECTROPHOTOMETER 21
FIGURE 1.7 FLOW DIAGRAM OF SCANNING ELECTRON MICROSCOPY 22
FIGURE 1.8 DIAGRAM OF BRUNAUER, EMMETT AND TELLER (BET)
ANALYZER 24
FIGURE 3.1 MAP INDICATING THE SAMPLE COLLECTION POINTS OF
HOSPITAL WASTEWATER 40
FIGURE 4.1 (A & B) THE IBP STANDARD OF 10µG ML-1 (A) ION
CHROMATOGRAM & MASS SPECTRA (B) AT FULL SCAN MODE 60
FIGURE 4.2 (A & B) RESPONSE IBP STANDARD PREPARED IN
DICHLOROMETHANE (A) AND METHANOL (B) 62
FIGURE 4.3 (A & B) RESPONSE IBP (10 µG ML-1) USING 1 µL
INJECTION VOLUME SPLIT MODE (1:50) (A) AND SPLITLESS MODE
(B) 63
FIGURE 4.4 (A) STRUCTURES OF THREE CEPHALOSPRINS;
CEFRADINE, CEFATOXIME AND CEFUROXIME 67
FIGURE 4.4 (B) SEPARATION OF THREE CEPHALOSPRINS; (1) CEFRADINE (2.753), (2)
CEFATOXIME (3.740) AND (3) CEFUROXIME
(4.533) USING 55%METHANOL+45% FORMICACID (0.05%) AT FLOW
RATE OF 1 ML MIN-1 AND MAX 260 NM 68
24
FIGURE 4.5 (A-B) ACID INDUCED HYDROLYSIS (1 M HCL) FOR 10
MINUTES AT 70 ºC CEFRADINE (A), CEFUROXIME (B) 70 FIGURE 4.5
(C) ACID INDUCED HYDROLYSIS (1 M HCL) FOR 10
MINUTES AT 70 ºC CEFATOXIME 71
FIGURE 4.6 RESPONSE AT ROOM TEMPERATURE AQUEOUS SOLUTION STABILITY OF
CEFRADINE, CEFUROXIME AND
CEFATOXIME 72
FIGURE 4.7 (A-C) RESIDUAL PLOT FOR REMOVAL OF OFLOXACIN
ONTO TREATED SAWDUST (A) HCL, NAOH (B) AND WATER (C) 80
FIGURE 4.8 (A-C) SHOWS THE EFFECT OF PH, CONCENTRATION,
AMOUNT OF SORBENT AND CONTACT TIME ON THE REMOVAL OF
OFLOXACIN 81
FIGURE 4.8 (A-C) MAIN EFFECT CHARTS FOR REMOVAL OF OFLOXACIN ONTO
TREATED SAWDUST (A) HCL, (B) NAOH AND (C)
WATER 83
FIGURE 4.9 HPLC CHROMATOGRAM FOR SPIKED HOSPITAL
WASTEWATER SAMPLE OFLOXACIN (10 µG ML-1) (A) AND AFTER
REMOVAL (B) 88
FIGURE 4.10 (A &B) RESIDUAL PLOTS OF CIPROFLOXACIN SORPTION
ONTO ACID TREATED PS (A) AND ALKALI TREATED PS (B) 93
FIGURE 4.10 (C&D) PARETO CHARTS FOR ACID TREATED PS (C) AND
ALKALI TREATED PS (D) 94
FIGURE 4.10 (E & F) RESPONSE SURFACE PLOTS FOR CIP SORPTION
ONTO ACID TREATED PS (E) AND ALKALI TREATED PS (F) 95
FIGURE 4.11 (A& B) SEM IMAGES OF ACID TREATED PEANUT SHELLS
AT 20 µM (A) AND 50 µM (B) 98
FIGURE 4.12 N2-ADSORPTION AND DESORPTION ISOTHERMS AT 77K
CORRESPONDING BJH PORE-SIZE DISTRIBUTION 99
FIGURE 4.13 HOSPITAL WASTEWATER SPIKED SAMPLE 20 µg mL-1 (A)
25
AND AFTER BATCH SORPTION RESPONSE (B) 100
FIGURE 4.14 (A) FTIR-ATR OF PREPARED HTC -ZM 102
FIGURE 4.14 (B & C) SEM IMAGES OF HTC-ZM AT 10µM (B) AND 5 µM
(C) 103
FIGURE 4.15 N2-ADSORPTION AND DESORPTION ISOTHERMS AT 77K
CORRESPONDING BJH PORE-SIZE DISTRIBUTION 103
FIGURE 4.16 (A & B) RESIDUAL PLOTS FOR (A) DICLOFENAC SODIUM
AND (B) IBUPROFEN SORPTION ONTO HTC-ZM 106
FIGURE 4.16 (C & D) MAIN EFFECT PLOT OF % SORPTION OF (C) DFS
AND (D) IBP ONTO HTC-ZM 107
FIGURE 4.16 (E &F) RESPONSE SURFACE PLOT FOR SORPTION OF IBP
(E) AND DFS (F) ONTO HTC-ZM 108
26
ABBREVIATIONS
ANOVA Analysis of variance
CCD Central composite
design
CIP Ciprofloxacin
DFS Diclofenac Sodium
DAD Diode array detector
D-R
Dubinin-
Radushkevich
Isotherm
EPA Environmental
protection agency
FT-IR Fourier transform-
infra red
GC/MS
Gas
chromatograph/mass
spectrometer
HPLC High performance
liquid chromatography
HLB
IBP
Hydrophilic lipophilic
balance
Ibiprofen
Kg Kilogram
KD-R D-R adsorption
capacity
KF Freundlich adsorption
capacity
27
LOD Limit of detection
LOQ Limit of quantification
M Molar
mg Milligram
mL Milliliter
µm Micron meter
µg Microgram
µl Microliter
NaOH Sodium Hydroxide
OFL
pH
ppb
ppm
Ofloxacin
Negative logarithm of hydrogen
ion concentration
Part Per Billion
Parts Per Million
PPCPs Pharmaceuticals and personal care
products
PhACs
Pharmaceutically active
compounds
rpm Rounds Per Minute
Qm Langmuir adsorption capacity
RSD Relative Standard Deviation
RSM Response Surface Methodology
SD Standard deviation
SPE Solid phase extraction
SEM Scanning electron microscopic
28
CHAPTER 1 INTRODUCTION
1.1 Emerging pollutants in water systems
The problem of contaminated water around the world has deeply
concerned the scientists. A broad range of substances are reported in the
literature as emerging pollutants (EPs) [1]. Various synthetic chemicals, for
example pesticides, cosmetics, pharmaceuticals and disinfectants etc. are used
in everyday life but these all chemicals are considered as EPs. Environmental
scientists have generally reported two main issues related to water; one is to
sustain water quality and other is to prevent water systems from contamination.
Presence of EPs in aquatic environment is well reported worldwide, whether
in bottled or tap water [2-5]. The progressive development in analytical
instrumentation has made it possible to determine EPs from aqueous samples
at trace levels [6-7].
Fate of EPs in environment has not been elucidated yet, therefore
environmental impacts of residues are unknown. The reported literature
confirms residual presence of the vast number of pharmaceuticals and personal
care products (PPCPs) in conventional wastewater treatment plants (WWTPs)
as well [8-9]. The WWTPs are the main source of these contaminants in the
environmental water systems due to their incomplete removal by precise
treatment methods [10-12]. Focused research on PPCPs began in Europe about
three decades ago, but during last few years an increase in research
publications show that the interest of researchers have increased worldwide
[13].
The continuous and unregulated releases of pharmaceutically active
compounds (PhACs) residues in the environmental water ways are often
detected. Non-stop and unregulated discharge of PhACs into aquatic
environment is causing adverse effects on aquatic life, which is alarming
situation [14-16].
29
1.2 Pharmaceutically active compounds in environmental water systems
Pharmaceuticals are manmade compounds use to prevent and treat
diseases [17]. After intake, some of the drugs are fully absorbed while others
may be excreted by body as metabolites or as parent compounds, consequently
released into environment [18]. The PhACs and their metabolites present in
water systems at very low but detectable concentrations. Majority of PhACs
and metabolites are not degraded or removed by conventional treatment plants
and as a result from wastewater effluent get transferred into environmental
water ways [19].
Although, PhACs remains in water at very low levels they are still considered
as potential contaminant [20]. The high risk of contamination has been
observed commonly in urban areas, where higher population results in increase
throughput of sewage discharge. In urban areas low surface water flow
synergizes contamination risk from sewage wastewater [21]. Sewage treatment
plants (STPs) are inefficient to treat the sewage wastewater completely.
Likewise, hospital wastewater treatment plants are not exception. [22-23]. Till
now the almost all water bodies like, rivers, ground, influent, effluent waters
and etc. are found contaminated with PhACs in the range of ng L-1 to µg L-1
[24-25].
1.3 Pharmaceutically active compounds as contaminant
As compared to other organic contaminants, PhACs were noticed only
lately by environmental scientists. Published scientific data about PhACs
presence in environmental samples has increased considerably [26-27].
Ground water, which is used for drinking purpose is threatened by the urban
municipal sewage discharge as compared to surface water flow and can be
easily contaminated by organic compounds. Hence a number of
pharmaceutically active compounds (PhACs) are not fully removed by the
sewage treatment plants (STPs) and enter into water ways [28-30].
Different water matrices for example; river, ground, sewage, etc. were studied
and the presence of PhACs in the range of ng L-1 to µg L-1 was observed. Even
30
though these PhACs are at low levels in water systems, they are causing
adverse effect on aquatic life, human beings and other living systems [31-33].
1.4 Persistence of pharmaceutically active compounds in water systems
It can be noted that pharmaceutical compounds with low excretion rates
(e.g., ibuprofen, carbamazepine, sulfamethoxazole, diclofenac and
primidione) are not necessarily present at low levels in the raw wastewater.
This is possibly because the low excretion rates are offset by the massive use
of these compounds. In addition, local common diseases can induce a higher
consumption of specific pharmaceuticals in certain periods. In literature it is
reported: some pharmaceutical compounds (acetaminophen, caffeine,
ibuprofen, naproxen and salicylic acid), one biocide (triclosan), one surfactant
(nonylphenol) exhibit relatively high concentrations. Generally, the
L-1) in
WWTP influent were ibuprofen, atenolol, caffeine and nonylphenol. For
instance, ibuprofen was the most abundant compound detected in the influent
of four WWTPs in Spain, with the concentration levels ranging from 12.13 to
L-1 [34-35].
The observed high levels of these drugs could be explained by high
consumption rate and easy accessibility from over the counter of these drugs.
Steroid hormones and pesticides generally show lower detected concentrations
L-1) as compared with compounds from other groups.
The concentrations of most micropollutants in effluent ranged from 0.001 to 1
L-1, which were one to two orders of magnitude lower than those in effluent.
Some abundant compounds in effluent were discharged at relatively high
concentrations. For instance, atenolol, caffeine, ibuprofen, naproxen,
nonylphenol and triclosan were detected in L-1
in treated effluent. In contrast, steroid hormones were found in wastewater at
much lower le L-1). However, their occurrence even at low
concentrations is a concern because of their high estrogenic effect [34, 36-37].
31
1.5 Classification of drugs
Drugs are classified according to chemical category of active ingredient or
their specific functional status. Each drug can be classified into one or more
drug classes. Drugs classification based on therapeutic utilization as under:
1. Antibiotic drugs: destroy the growth of bacteria
2. Analgesics: reducing pain (painkillers)
3. Antipyretics: reducing fever (pyrexia/pyresis)
4. Antimalarial drugs: use to prevent from malaria
5. Antiseptic drugs: to stop microbial infection.
Antibiotics and analgesics are most commonly used drugs, which also persist
in environment, are discussed below:
1.5.1 Antibiotics: This constitutes various groups of compounds which are
used to treat bacterial infections. The frequently prescribed groups of
antibiotics for treatment of human and veterinary curing medicines are as
follows: sulfonamides (SAs), aminoglycosides (AGs), macrolides (MCs),
- -LCs), tetracyclines
(TCs), amphenolicols (AMPs), glycopeptides (GPs), polyether ionophores
(IPhs) and lincosamides (LCs) [38]. -lactams; penicillins and cephalosporins
accounts for nearly 50-70% of antibiotics consumption in EU and USA etc.
Antibiotics are multi-group compounds and their determination in complex
samples like environmental waters, milk and serum samples have been subject
of interest to analytical and environmental chemist [39-40]. Several
chromatographic assays, especially LC and CE coupled to MS or tandem MS
are reported for screening of drugs in wastewater samples [41-43].
It is reported that extreme presence of antibiotics are penetrating the
environment as compared to other classes of drugs [44-45].
The rate of metabolism of antibiotics is observed to be partial or incomplete in
human bodies as well as in case of other animals. Antibiotics excreted mostly
unchanged (10-90% unmetabolized) from body through waste products like
urine and feces. Moreover antibiotics/metabolites show resistant to
32
degradation and persistent to environment and become cause of contamination.
In addition, it is assumed that the chronic exposure to antibiotics could induce
the development of antibiotic-resistant pathogens in environment [46-49].
1.5.2 Analgesics: Analgesics are used to relieve the symptoms of pain. These
can be classified in three groups: 1) Non-narcotic drugs, like aspirin and
paracetamol, commonly used for their antipyretic and analgesic properties; 2)
Narcotic analgesic drugs, morphine is the most common type in this class. The
narcotic analgesics are used for post-operative and cardiac pain and also for
relieving pain of terminal cancer; 3) Non-steroidal anti-inflammatory drugs
(NSAIDs), such as ibuprofen (IBP), naproxen (NPX), diclofenac (DFC) and
ketoprofen (KFN). These show analgesic as well as antipyretic properties[50].
Owing to their dual action, the consumption ratio has increased worldwide.
Their frequent utilization by human beings, and partially metabolized
excretion or dumping as unutilized form, produces adverse impact on aquatic
environment. Their concentration at lower range in ng L-1 µg L-1 is frequently
detectable in water ways [51-52]. Polar molecules with low adsorption
coefficients likeanti-inflammatory drugs tend to remain in the aqueous phase
favoring their mobility, therefore, reaching the aquatic environment [27-29].
1.6 Sources of pharmaceutically active compounds in water systems
Figure 1.1 [53] shows overview of various contaminating sources for
PPCPs, which includes unmatabolized parent drugs, disposal of unused drugs,
underground leakage from sewage system, hospital waste and disposal from
drug laboratories etc. descriptive legends of Figure 1.1 are give in Table 1.1.It
may be pointed out that among various sources hospital wastewater is the
leading source for contamination of PhACs.
33
Figure 1.1 Origins and fate of PPCPs in the environment
Table 1.1 Origins and fate of PPCPs in the environment description of legends given
in figure1.1
Legends
1) Usage by individuals (1a) and pets (1b):
Metabolic excretion (unmetabolized parent
drug, parent-drug conjugates, and
bioactivemetabolites); sweat and vomits,
Excretion exacerbated by disease and slow
dissolving medications
Disposal of unused/outdated medications to
sewage systems
Underground leakage from sewage system
infrastructure
Disposal of euthanized/medicated animal
carcasses serving as food for scavengers (1 c)
6) Discharge of regulated/controlled industrial manufacturing waste streams
34
2) Release of treated /untreated hospital
wastes to domestic sewage systems (weighted
toward acutely toxic drugs and diagnostic
agents, as opposed to long-term
7) Disposal/release from clandestine
drug labs and illicit drug usage
Disposal to landfills via domestic
refuse, medical wastes and other hazardous wastes Leaching from defective (poorly engineered )
landfills and cemeteries
medications ); also disposal by pharmacies,
physicians, humanitarian drug surplus
Release to private septic/leach fields
3) Treated effluent from domestic sewage
treatment plants discharge to surface waters
or re-injected into aquifers (recharge)
Overflow of untreated sewage from storm
events and system failures directly to surface
waters
8) Release to open waters from aquaculture
(medicated feed and resulting excreta)
Future potential for release from molecular
pharming (production of therapeutics in
crops)
sewage discharged directly to surface waters)
Release from agriculture: spray drift from tree
crops (e.g.,antibiotics)
Drug from medicated domestic animals (e.g.,
feed)-CAFOs (confined animal feeding
operations)
9) Release of drugs that serve double duty as
pest control agents:
35
5) Direct release to open waters via
washing/bathing/swimming
10) Ultimate environmental transport/fate;
most PPCPs eventually transported from
terrestrial domain to aqueous domain
phototransformation (both direct and indirect
reactions via UVlight) volatilization (mainly
certain anesthetics, fragrances) some uptake
by plants
repairable particulates
containing
sorbed drugs (e.g., medicated-feed dusts)
36
1.7 Impact of pharmaceutically active compounds on human health and
aquatic environment
The varied occurrence of PhACs in different environmental systems raises
concerns about their potential harm to ecosystem and human health. Regarding
their environmental concentration levels, toxicity of PhACs is not sighted abruptly.
Although, reported by Lindsay et.al. the residue of veterinary diclofenac affected
the significant decline of vulture population in Pakistan [54].
Antibiotics continuous disclosure to environment is presume to cause the antibiotic
resistance genes (ARGs) which lead to potential damage for ecosystem [55-56].
Antibiotic resistance genes encoding resistance to a broad range of antibiotic
species, such as macrolides, sulfonamides, fluoroquinolones, and tetracyclines,
occur ubiquitously in hospital and livestock feeding effluents, municipal
wastewater, surface water, as well as drinking water resources [56-57]. On the other
hand, multiple antibiotic resistant (MAR) superintegrons, which may contain over
100 ARG cassettes, have been also discovered [57]. Adverse effect of few selected
PhACs is given in Table 1.2 [58]. It may be noticed that the persistence of PhACs
can inhibit the growth of algae, may develop oxidative stress and can induce
endocrine disruption.
Table 1.2 Adverse effects of PhACs
Pharmaceutical Compounds Concentration
(µg L-1)
Exposure
Span(days)
Adverse effect Reference
Roxithromycin, Clarithromycin,
Tylosin
40-64 3 Growth inhibition of
algae
14
Caffeine 2000 7 Endocrine disrupt 23
Diclofenac 5 28 Renal lesions
Carbamazepine 200 42 Oxidation stress of rainbow
trout
28
Gemfibrozil 4420 1 Growth inhibition of
algae
Propranolol 0.5 28 Oxidation stress of
goldfish
10
Triclosan and triclocarban 0.4-10 3 Growth inhibition of
algae
47
37
Risk assessments of PhACs can be calculated using recommended guidelines by
European Medicines Agency (EMA), in which the risk quotient is calculated as the
ratio between predicted environmental concentrations (PEC) and predicted noeffect
concentrations (PNEC).
adverse ecological effects [59-60]. A prioritization approach based on number of
prescriptions and toxicity information as well as human metabolism and
wastewater treatment removal information was performed on 200 most-prescribed
drugs in U.S. in 2009 [61] for the risk assessment of pharmaceuticals were found
to be levothyroxine montelukast received the maximum risk score.
Commonly on the basis of current environmental contamination levels of PhACs,
it is believed to be low possibility of extreme toxicity; however, chronic toxicity of
these PhACs cannot be underestimated.
1.8. Removal technologies for persistent pharmaceutically active
compounds in water systems
The majority of PhACs are not completely metabolized in patient body, and
as an outcome some of the parent compounds along with metabolites are excreted
[62].
Pharmaceuticals are not only used to combat human and animal diseases but are
widely used in farming and aquaculture. Even though the concentration of single
drug in the aquatic environment may be in the micropollutant range (sub-parts per
billion), the presence of numerous drugs sharing a specific mode of action could
lead to additive exposures that could cause significant effects [63].
The organic micro pollutants, like dyes or fragrances and anthroprogenic
contaminants are typically treated with ozonation process [64]. The oxidation with
ozone series at high reaction rates about 106 L /mol s for PhACs for example
diclofenac (secondary amine), carbamazepine (double bond), roxithromycin
(amine), and sulfamethaxzole (amine). Till today information about PhACs in
environment and their further fate after exposure need to be explored to understand
the after effects [65-67].
38
Adsorption techniques are remarkable among various technologies studied for
removal of persistent compounds. However, finding specific adsorbent for selected
analyte, extensive study is required to characterize the chemistry behind the
phenomenon [68-69].
Adsorption is one of the most widely applied techniques for pollutant removal from
contaminated media. Common sorbents include activated carbon, molecular sieves,
polymeric adsorbents, and other low-cost materials [70].
Only few studies have been carried out for decontamination of PhACs from
aqueous solutions using sorptive technologies [71-74]. Organic waste
materials/biomasses have been found efficient for decontamination of toxins.
Adsorptive removal of PhACs using organic materials like pomegranate peel, black
tea leaves and cork are reported [75-76]. For the remediation of metal ions from
water system sawdust and peanut shells are reported earlier but not exactly for our
selected PhACs. Sawdust and peanut shells are environmentally friendly
biosorbents both after treatments are good option for remediation of PhACs. All
the three sorbents reported so far showed affinity towards sorption of PhACs;
however, systematic optimization for optimal efficiency is not worked out [7778].
In addition to these types of bio-based sorbents, a new generation of hydrothermal
carbonized (HTC) biomass has evolved as sorbent in recent years [79-80].
The key of interest is in synthesis of HTC material where properties of material
like particle size, functionality and shape can be easily tuned for specific
application [81].
Carboneous materials production from biomaterials has potential to produce wide
variety of carbonic products. In 1913, Bergius and Specht first time reported about
hydrothermal transformation of cellulose into coallike products. Further
development in same technique has evolved a new field in material chemistry
[8284].
39
Organic matter like biomass decompose upon heating in absence of oxygen, which
is termed as thermochemical decomposition, also known as pyrolysis [85]. If this
thermochemical decomposition take place in aqueous environment, at subcritical
temperature, it is called hydrothermal carbonization (HTC) or hydrous pyrolysis.
Benefits of HTC products include conversion of wet biomass into carboneous
material without any vigorous drying in whole process and heating at low
temperatures as compare to dry pyrolysis. During the hydrothermal process
carbonization of carbon materials (pure like glucose, and complex like biomass)
take place at relatively low temperatures (160-250 °C). During the process very
small amount of gas is produced (1-5%), where pressure of around 20 bar is
observed. The conditions are favorable to convert the source organics into HTC
products [86-87].
Carbonisation takes place in water under autogeneous pressures thus avoiding
precursors drying costs. Hydrothermally carbonized biomass (hydrochar) has
received increased attention recently as a potential agent for contaminant
remediation and soil improvement [88]. However, the study suggests great
potential for natural sorbents for removal of PhACs, therefore opens the door for
exploration of this type of materials.
1.9. Sorption studies
Sorption is one of the most widely applied techniques for pollutant removal
from contaminated media. The transfer of molecules from an aqueous phase to a
solid phase, such as soil or sediment is referred to as sorption and the reverse
process is desorption. Adsorption is the transfer to a two dimensional surface and
absorption is the dissolution into a three dimensional matrix. Sorption is key
process in many chemical phenomenons, where density of sorbate changes visibly
from the environment after sorption process [89].
Sorption is most used technique for transfer of contaminants from one phase to
another. Fundamentally sorption is a surface phenomenon, which involves or is at
40
large extent govern by physical interactions. In some cases along with physical
interaction, chemical interactions may also take place [90].
Asdorption phenomenon can be studied and characterized by various isotherms.
The most commonly used isotherms for the application of sorption equilibrium
studies in water and wastewater treatment are the Langmuir, Freundlich and
Dubinin Radushkevich isotherms
1.9.1. Langmuir adsorption isotherm
Langmuir adsorption isotherm is used to quantitatively determine adsorbate
monolayer on the surface of adsorbent. Langmuir isotherm allows the study of
equilibrium between adsorbent surface and adsorbate amount in liquid phase. The
linearized of Langmuir adsorption isotherm is given in Equation 1.1.
Equation 1.1
In above equation qe (µg g-1) represents sorbate amount at equilibrium in solid
phase; Ce (mg L-1) sorbate amount at equilibrium in liquid phase; qmax is maximum
adsorption capacity and KL is Langmuir isotherm constant (L mg-1).
1.9.2 Freundlich adsorption isotherm
Heterogeneous surfaces are well studied by Freundlich isotherms. The data of
adsorption study quantitatively is given by below equation. In given Equation 1.2
qe (mg g-1) is amount adsorbed per gram of adsorbent at equilibrium; Ce (mg L-1)
sorbate amount at equilibrium in liquid phase; Kf (L g-1) is measure of adsorption
capacity and n (dimensionless) is heterogeneity factor represents Freundlich
constant, intensity of adsorption.
Equation 1.2
41
1.9.3 Dubinin Radushkevich adsorption isotherm
Detailed study of heterogeneous surface can be done by studying its Gaussian
energy distribution quantitatively as proposed by Dubinin-Radushkevich (D-R)
isotherm. The empirical equation for this isotherm is given below (1.3). KD-R is
theoretical maximum adsorption capacity (mol g-1); qe is amount of adsorbate in
the adsorbent at equilibrium (mol g-1); = Polanyi potential and is equal to RT ln
(1+1/Ce), T is temperature and R is universal gas constant; (mol2 k-1j-2) is mean free
energy of adsorption per mole of the adsorbent [91].
Equation 1.3
1.10 Mathematical and statistical approach for removal technologies
Typically, sorption studies are performed by optimizing single variable at a
time which results in number of experiments. Multi-variant optimization using
statistical approaches are nowadays common practice to reduce number of
experiments without compromising the quality of data. Response surface
methodology (RSM) is an experimental design which helps to optimize the process.
Using this approach, factors which may affect the adsorption (any other process)
are selected like pH, amount of sorbate, contact time etc., afterwards these factors
are coded at minimum and maximum levels. Usually the coding is -1, 0, +1, where
-1 is lowest and +1 is highest value of each factor. RSM is a three steps process;
initially the statistical data of experiment is generated, in second step the evaluation
of coefficient are obtained by mathematical model. At final step, validation of
proposed model is calculated which is obtained by optimum conditions for
maximum outcome [92].
RSM uses many designs like central composite design (CCD) -bekhen
at different levels etc. In this study multi-variant sorption
42
optimization was performed by using Draper-Lin small composite design which
belongs to CCD model. The design contained eighteen batch experiments; each
experiment is conduct at the different level of independent variables.
1.10.1 Central composite designs (CCD)
The statistical approach of central composite design (CCD) is the frequently used
design for multi-variant process optimization. This design functions at three levels
of each selected variable as -1, 0 and 1 factorial level. It is commonly used due to
its economical compatibility with experimental subset of three levels of design. To
achieve the desirable properties of design the value of and
number of centre point of replications are chosen accordingly in CCD [93] .
Centerpoints is the number of experiments to be added to the base design, which
are additional experimental runs located at a point midway between the low and
high level of all the factors. Each additional centerpoint adds one degree of freedom
from which to estimate experimental error.
Axial parameter is positioning of the centerpoints with respect to the runs in the
base design. They may be randomly scattered throughout the other experimental
runs, spaced evenly throughout the other runs, or placed at the beginning or end of
the experiment. The first two options are usually preferable. In this study,
randomized approach for design with eighteen set of experiment was used for
optimization.
1.11 Current situation of PhACs in Pakistan
The pharmaceutical industry started growing soon after the creation of
Pakistan in 1947. The industry established strong foundation all along with
multinational pharmaceutical companies. The multinational companies provide the
financial support as well as advancement to this field for locals to provide them
quality medication [94].
, the basic active chemical ingredients are
43
imported and further formulation is processed. The wide spectrums of drugs are
formulated like analgesics, antibiotics, anti-clotting agents, etc. The
pharmaceuticals compounds are formulated from curing diseases to multivitamin,
and other tonics in the form of tablets, capsules, ampoules, syrups, etc.
[95]
In hospitals and households, pharmaceuticals such as antibiotics, antidepressants,
analgesics, and hormones are used on a regular basis. After consumption or
application, many drugs are non-metabolized and excreted into wastewater.
There are only few or almost no studies on existence of PhACs in water streams of
Pakistan. It can be concluded from the reported literature the possibility of these
PhACs in our water systems maybe higher as there are no proper regulations for
drug circulation and treatment for wastewater streams in Pakistan.
A detailed study non-targeted screening of water streams near Hyderabad, Pakistan
was published by Shaikh etal [96]. Ibuprofen along with a large number of other
organic contaminants was identified at low µg mL-1 levels.
There are number of pharmaceutical companies producing various drugs in bulk
quantities, which are massively consumed by Pakistani public. There is no
authentic consumption data of drugs available to public.
1.12 Determination techniques for pharmaceutically active compounds
in water system
There is also a growing need for analytical methods that could detect low
levels of parent compounds and their biodegradation products. Chromatographic
methods are a good choice for such multicomponent samples. A multi-residue
analytical method has been developed and validated for determining a multi class
of pharmaceuticals: the anti-epileptic carbamazepine, seven
analgesic/antiinflammatory drugs (mefenamic acid, indomethacine, ibuprofen,
naproxen, diclofenac, ketorolac and acetaminophen), the analgesic opiate codeine,
two
-blockers (atenolol and propranolol),
44
antibiotic (trimethoprim, metronidazole, and erythromycin) and the anti-ulcer
ranitidine in hospital effluent wastewaters. The method allows simultaneous
extraction of the pharmaceuticals compounds by solid-phase extraction (SPE) using
the Waters Oasis HLB at pH 7 [97-99].
Since most frequently employed technique is chromatography with SPE, and also
employed in our work, a brief description on techniques are discussed in next
header.
1.13 Instrumentation
1.13.1 High performance liquid chromatography ( HPLC)
The most popular physical separation technique is high performance liquid
chromatography (HPLC). The separation is attained in HPLC between two phases,
a mobile phase (liquid phase) and stationary phase (packed column). The analyte
distribution is evaluated qualitatively and quantitatively.
An HPLC instrument is consists of a pump, column, injector, detector and data
processing device (Figure1.2).
HPLC instrument pumps operational pressure ranges from 2000psi to 6000 psi in
regular analytical process.
Figure 1.2 Block diagram of HPLC instrument
45
The pump is one of the running components of HPLC. Pump helps to run the
solvents and anlytes through the column. On the basis of pumps operating
system there are most commonly three types: syringe type pump, constant
pressure pump and reciprocating piston pump. HPLC instruments exist with
one to four pumps depending on application.
Sample input can be conduct in HPLC by two ways 1) Manual injection
(Rheodyne or valco) and 2) Automatic injection. The traditional manual
injection port is designed with six port rotator valves. Constant volume loop
of rheodyne injector take sample at atmospheric temperature with manual
syringe. Hence in automatic injector the software program runs the analyte
from sample loop to column.
The selection of detector is based on the desired analytes nature. Following
are the detectors use: 1) Ultra violet/Visible, 2) Photo Diode array, 3)
Refractive Index 4) Evaporating light scattering, 5) Conductivity, 6)
Fluoresence, 7) Optical rotation, 8) Chemiluminesence, 9) Muti angle laser
light scattering and 10) Mass spectrometer.
The porous particles filled HPLC columns helps to separate the anlytes.
Column carrying the mobile phase along with sample through specific
choice of column according to desired separation process by analyst.
Variety of commercial columns are available by manufacturers; for example
C-18, C-8, C-4 and CN, etc. to produce the required separation.
For data processing HPLC connected to a computer with workable software.
The software regulates the pump, detector and injectors function as required
[100].
1.13.2 Liquid chromatography mass spectrometry (LC/MS)
and characterize the chemical components with more depth. HPLC coupled with
mass spectrometer (MS) as shown in Figure 1.3 is a technique in which common
HPLC detector replaced with MS. It is very useful technique for organic molecules
and their metabolites detection. MS as a detector gives direct and insightful
46
structural information of organic compounds as proteins, pharmaceuticals and
paints etc. Usually for the analysis of multi component samples LC/MS utilized,
every component initially separated by LC then further detected with MS.
LC and MS interface improvement is centre of attention for scientist and
manufactures. LC uses liquid phase transport with high pressure from column to
achieve the separation of eluate of sample; Whereas MS operates at vacuum with
limited pressure. LC easily separates analyte with inorganic buffers but MS requires
volatile buffer system. Additionally, most LC systems run at ambient temperature
while MS works at elevated temperatures [101].
Figure1.3 Block diagram of LC/MS
1.13.3 Gas chromatography mass spectrometry (GC/MS)
Gas chromatography (GC) is a technique in which a gas acts as mobile phase to
transport volatile sample to the capillary column for detection. GC coupled with
mass spectrometry (MS) is a sensitive technique. GC/MS is frequently use in the
fields of research and chemical industries for analysis of volatile organic
compounds. The main parts of GC/MS (Figure 1.4) instruments are as follows: 1.
Ionization source, 2. Mass analyzer, 3.Detector, 4.Vacuum system and
47
5.Computer with operating software.
Figure 1.4 GC/MS instrument block diagram
1. Ionization source use to inonized the sample into product ions. The intensity
of ionization source selected on the basis of desired samples analysis. Usually
universal ionization source is electron impact (EI) ionization.
2. Mass analyzer works as filter, it helps to separate the ions produce by ion
source with the mass to charge ratio of product ions.
3. Detector works to convert the mass analyzers output ion beam in to readable
signal by data recorder.
4. Vacuum system requires processing the mass analyzer, high levels of
vacuum needed to measure the mass to charge ration of ionized ions flawlessly.
5. Computer is coupled with instrument to run designed software to control
and manage analysis process [102].
1.13.4 Solid phase extraction (SPE)
Sample preparation is one the most tedious and challenging task in analysis. Solid
phase extraction (SPE) is a frequently use technique for preparation of aqueous
48
samples (Figure 1.5). It is a versatile technique use in pharmaceuticals, paints,
biological fluids, environmental samples etc. Solid phase employed during sample
preparation not only extracts the anlyte of interest but also do the following:
Figure 1.5 Steps of solid-phase extraction for analyte
1.13.5 Ultraviolet/visible spectroscopy
Instruments for measuring the absorption of ultraviolet or visible radiation (Figure
1.6) are made up of the following components;
49
1. Radiation sources, which generates a broad band of electromagnetic radiation
(UV and visible). A good spectrometric source should have a stable, high
intensity output that covers a wide range of wavelengths.
2. Wavelength selector (monochromator) a dispersion device which selects a
particular wavelength or wavelengths.
3. Sample containers must contain the liquid or solution in the spectrophotometer
sample area; which is placed in the sample beam, a cell becomes an active
optical component. All UV spectra can be recorded in the solution phase and
the samples are placed in cells or cuvettes. Cells may be made of glass, plastic
or quartz.
4. Detector is a device that convert radiant energy into electrical signal, One or
more detectors use to measure the intensity of radiation. Detector should be
sensitive with efficient response rate, they are classified into four basic
categories:
a) Phototube
b) Photomultiplier tube
c) Diode array detector
d) Charge coupled devices
5. Signal processor and readout helps to convert output current into readable
signal with help of galvanometer or microammeter. The meter is calibrated in
terms of transmittance along with optical density [104].
Radiation
source
Monochromator
Cuvette
Signal
processor
Detector
Figure 1.6 Block diagram of UV/visible spectrophotometer
1.13.6 Scanning electron microscope (SEM)
50
Scanning electron microscope (SEM) is in which, focused beam of high-energy
electron uses to generate a divers signals from the surface of solid samples to collect
comprehensive surface analysis. Major components of SEM (Figure 1.7) are as
follows:
1. Electron source is used to produce electrons by thermionic heat and accelerated
with voltage from 1-40 kV into a narrow beamed at sample for imaging.
Commonly three types of electron sources are used for SEM analysis:
a) Tungsten (W) electron filament
b) Solid state crystal (CeB6 / LaB6)
c) Field emission gun
2. Lenses are use to focus the electron beam as it travels from source towards
down the column.
3. Scanning coils are used to deflect the beam of electrons in X and Y axes of
sample [105].
51
Figure 1.7 Flow diagram of scanning electron microscopy
4. Sample chamber contains translation stage, tilt and rotation devices,
temperature stages, optical cameras and variety of gadgets to assist the sample
imaging.
5. Detector is use to collect the electrons coming from the sample surface, two
types of detectors use for SEM imaging: a) secondary electron detector and b)
backscattered electron detector [105].
1.13.7 Brunauer, Emmett and Teller (BET) analyzer
It is used for surface area and porosity analyses to measure the specific surface area,
isotherms, pore size and pore size distribution of analyte; major components of its
are as follows:
52
1. Pumps are use to maintain accurate flow rate of gases and to sustain vacuum
of system throughout analysis.
2. Sample ports of two types are present (a) samples pretreatment (degassing)
and (b) samples analysis, number of ports vary from four to six commonly in
instrument.
3. Sample cells (quartz) are typically comes in three sizes (a) 6 mm, (b) 9 mm
and (c) 12 mm; a minimum 0.05 gm of amount of sample required to determine
surface area.
4. Dewar, isothermal jacket filled with liquid nitrogen used to control the
temperature of sample cell during analysis.
5. Injection port is used to inject the adsorbate gas into sample cell.
6. Four types of detectors are utilized for BET analysis;
(a) Thermal conductivity detector (TCD)
(b) Temperature programmed detector (TPD)
(c) Temperature programmed reduction (TRD)
(d) Temperature programmed oxidation (TPO)
7. Data acquisition with the help of software efficiently generates reports for
adsorption and desorption of analyte [105].
53
Figure 1.8 Diagram of Brunauer, Emmett and Teller (BET) analyzer
1.14 Aims and objectives of current study
This study is aimed at collecting data for use of various pharmaceuticals in
hospitals of Hyderabad, Pakistan and to evaluate their possible existence in hospital
wastewaters.
Following objectives were set to achieve the aims of the study:
1. Surveying local hospitals to identify frequent prescribed pharmaceuticals.
2. Thorough monitoring of hospital wastewater for detailed screening of PhACs.
3. Selection of PhACs on the basis of above study and studies reported in
literature.
4. Utilization of modified natural adsorbents for removal of selected PhACs.
5. Performance of adsorption experiments to estimate adsorption capacity,
kinetics of selected natural adsorbents.
1.14.1 Output of research study
Database for pharmaceutical compounds in hospital wastewater status in Pakistan
(selected locations only).
Treatment options for PhACs removal will be available.
54
1.15 Structure of thesis
Chapter 1 comprises the introductory background of PhACs as contaminant
in water system and studies carried out about it. Brief discussion of used
techniques for this thesis followed by aims and objects of study.
Chapter 2 includes background of PhACs as contaminant and emphasis on
selected PhACs reported literature for determination and removal studies
status worldwide and specifically in Pakistan.
Chapter 3 has information about chemicals and solvents used for this study. It
also mentions the approach and techniques use for both determination and
removal of selected PhACs.
Chapter 4 contains detailed discussion of obtained results from developed
chromatographic methods along with screened data for selected PhACs in
our wastewater. It further gives comprehensive information about removal of
selected PhACs by treated natural sorbents.
Chapter 5 has conclusive remarks from this study with applicable
recommendations to prevent water ways to be contaminated from PhACs.
CHAPTER 2
LITERATURE REVIEW
55
2.1 Worldwide situation of PhACs in aquatic environment
Pharmaceuticals and personal care products (PPCPs) are consumed in the
households, human and veterinary medical care centers, and agricultural
applications. These products are consumed directly as purchased. PPCPs represent
a diverse range of chemical compounds. These PPCPs are potential contaminants
to aquatic environment [106].
Reports on the presence of human and veterinary pharmaceuticals compounds in
the environment started to appear nearly three decades ago [107-109].
PPCPs; the compounds which are used on daily basis turned out to be pollutant for
environment. Promising risks connected with releases of pharmaceuticals into the
environment have become an even more important issue for environmental
researchers and concern authorities [110-111]. The pharmaceutical compounds
with variety of therapeutic classes are consumed worldwide annually in large
number for prevention and treatment of diseases [112-113].
Pharmaceutically active compounds (PhACs) have been repeatedly detected in the
environment. However, not a single source is responsible for PhACs release. The
main contributing sources for these PhACs release to the wastewater (WW) have
been typically medically care units.
However, till present studies have shown that the input of PhACs via hospitals to
the environment is to a certain extent [114].
The pioneering evidence in the scientific literature about PhACs in treated
[115]. Earlier the analgesic like, clofibric
acid was detected in the range of 0.8-2 µgL-1 in the U.S.A. Soon after this PhACs
were found in U.K. up to 1µgL-1 same observation was found in Canada too [116].
The PhACs reporting is increased even their presence at lower level with the help
of advancement in analytical detection methods. It is clear that releasing
pharmaceuticals in the environment are gradually emerging as persistent
contaminant for water systems [117].
56
Up to date literature reported a broad range of pharmaceuticals from different
classes of drug and their metabolites (anti-inflammatory, beta-blockers,
sympathomimetics, antiepileptic, lipid regulators, antibiotics, etc.), approximately
more than 100 PhACs detected in water systems [118].
There are quite a lot of direct and indirect routes through which PhACs can be
entered into the water systems. Inadequately treated effluent from pharmaceutical
industries, hospital wastewater and municipal wastewater discharge are identified
as the major paths responsible for surface water contamination with PhACs
[119120]. The common conventional methods of treatment (i.e., biological,
physical, and chemical methods) have some degree of efficiency to remove PhACs.
Commonly wastewater treatment plants (WWTPs) proceed only two treatment
steps (physical and biological) while few of developing countries use a tertiary
treatment or an advanced sewage treatment (e.g. ultrafiltration, flocculation,
ozonation, advanced oxidation, or osmosis) [121-123]. Hospital waste poses a
significant impact on health and environment. The municipal wastewaters, hospital
wastewater and industrial wastewater carrying PhACs are consecutively causing
contamination of waters systems. At the time of treatment process of the municipal
wastewater, hospital wastewater and industrial wastewater, the variety of organic
compounds exist in these wastewater streams. Some of the PhACs have endocrine
disrupting behavior, even after processing at STPs they cope to escape in to
environmental water systems [124-129].
In hospitals a large variety of substances (e.g pharmaceuticals, radionuclides,
solvents and disinfectants) are used for medical treatment, and other purposes such
as diagnosis and research. Pharmaceuticals and their metabolites excreted by
patients, primarily in the urine, along with used diagnostic agents and disinfectants
are disposed of in wastewater [130-131].
Once a drug is disposed or excreted (along with its metabolites), it passes dissolved
or suspended in sewage to engineered sewage treatment facilities, to septic
facilities, leach fields, or directly into receiving waters (e.g., via illegal privies or
57
"straight-piping"); straight-piping serves to maximize the availability to the
environment of any PPCPs that are present since no treatment is used to remove
residues. Raw, untreated sewage can also enter the environment from sewage
distribution and treatment systems as a result of storm events (overflows), system
failures, and overcapacity; this is a common problem in those locales with aging
infrastructures or rapidly expanding populations [132-133].
The PhACs and their metabolites present in water systems at very low, but
detectable concentrations. The entrance of these PhACs initially take place from
patient excretion, which run into WWTPs. Majority of PhACs and metabolites are
not degraded or remove by conventional treatment plants and as a result from
wastewater effluent transfer into environmental water ways[134-136].
Pakistan is facing shortage of consumable quality water because of current
droughts and its increasing use for agriculture, domestic and industrial purposes.
Also there is no efficient wastewater treatment system available in country.
population is around 192,014,470 (April 2016 est.) and is
expected to grow approximately 250 million by the year 2025. With increasing
population use of water is also expected to increase for routine domestic,
agricultural and industrial purposes. Approximately 400,000m3 of domestic
wastewater day-1 is dropped off into canals. It is also reported that around 64% of
whole wastewater in Pakistan is drained without any treatment directly into rivers
or Arabian Sea. This continuous infusing of untreated and incomplete treated
wastewater is causing depletion of consuming water quality of Pakistan. Number
of reports observed the ground water and surface water have decline in the quality
[137]. Hence, it is need of time to prevent the natural water resource from depletion.
In many areas of country Farmers have no choice but to use wastewater for
cultivation purpose. Crops cultivated in wastewater may bring health hazard and
side effects [138-139].
58
In one report the diclofenac and five of its transformation products were found in
different water samples of Karachi, Pakistan in quite higher amount in microgram-
per-liter range [140].
2.3 Effects of PhACs on living being
Pharmaceuticals are manmade compounds to prevent and treat diseases.
These compounds are synthesized to cure and treat health threatening components;
the active pharmaceutical ingredients (APIs) in these compounds as parent
compound or its metabolites can release from consumer to environment [141].
Numbers of reported literature monitor these PhACs in environment via national
monitoring programs; they carry out the scientific research work, exposure and
remediation of these contaminants in ecosystem [142-143].
The alarming situation was observed for the environmental contamination with
PhACs, which include non-steroidal anti-inflammatory drugs (NSAIDs) as the
most consumed group of drugs worldwide. The nature of these NSAIDs observed
to survive from WWTPs and can persist in environment. The high rate of
environment [144-146].
In one scientific report the direct correlation between residues of diclofenac and
renal failure was examined, the diclofenac treated livestock which were later
consumed by vultures confirmed the adverse impact of diclofenac on vulture
population. It was concluded that the residues of diclofenac were main reason for
the vulture population decline in Pakistan and India [147].
2.4 Analytical methodology of selected
pharmaceutical compounds
Analytical methods with more precise and good sensitivity are needed to
investigate residual contaminants in environment. Residual contaminants are
usually detected and determined by atmospheric pressure ionization of HPLC/MS
in variety of water matrices. With specific sample treatment GC/MS is also used
59
for qualitative and quantitative analysis of PhACs residues in waters. Sample
preparation by liquid-liquid extraction, solid-phase extraction and microextraction
are very common. Both online and off-line sample preparation methods are
reported in literature [148-151].
To carry out the hospital wastewater studies for PhACs of Hyderabad, Sindh,
Pakistan hospitals, two persistent classes of drugs; antibiotics and analgesics, which
are frequently prescribed drugs were surveyed (Table 2.1).
Table 2.1 Selected pharmaceutical compounds and their structure
Drug Name Structre
60
2.4.1 Antibiotics
Antibiotics are mostly prescribed drugs in hospitals, for curing infections or
treating bacterial diseases [152-153]. As the result of insufficient hospital
wastewater treatment, antibiotics have been detectable in aqueous environmental
samples.
Analgesics
61
Compared to other persistent PhACs, antibiotics are centre of attention because of
their potential behavior of bacterial resistance effect. The nonstop releases of
antibiotics are causing the hazardous effect on aquatic life too [154-156].
Therefore, in recent years, antibiotics have been measured as potential emerging
environmental pollutant, most reports show multitude of antibiotics and their
metabolites survive WWTPs and are found in surface aquatic samples [55,
157158].
Quite high concentrations and frequencies of antibiotics were observed in hospital
wastewater as compare to other contributing points. Selected antibiotic drugs are
listed in Table 2.1, their detected quantities in last five years are shown in Table
2.2.
2.4.2 Analgesics and anti-inflammatory drugs
Presence of persistence of these PhACs in environment is rising day by day. The
drugs are pain comforters, antipyretic and inflammation reducers and most
common pharmaceutical compounds used in hospitals as well as in households
[50]. After antibiotics reported literature the analgesics and non-steroidal
antiinflammatory (NSAID) drugs are the most contaminant of concern in the
environment. Their widespread appearance in the aquatic environment is because
of their high consumption and their incomplete removal during wastewater
treatment [159-160].
The commonly used NSAIDs are ibuprofen, naproxen and diclofenac; These
NSAID and their metabolites are frequently observed in aqueous environmental
samples [161-163].
In reported literature diclofenac (DFS) was repeatedly observed in wastewater up
to ug/L level, however lower quantities were found in surface water. The same
behavior was observed for ibuprofen (IBP) [164-166].
IBP is very frequently prescribed and is often detected in water systems
62
along with disposal of unused IBP. Concentration of IBP detected in effluents
ranges from 10 ng L-1 to 169 µg L-1 [6, 167-168]. The cefradine, cefuroxime,
cefotaxime, ciprofloxacin, ofloxacin, diclofenac and ibuprofen were detected in
hospital wastewater from 2011-2015 are shown in Table 2.2.
Table 2.2 Selected pharmaceutical compounds detection in hospital wastewater
reported in year 2011-2015
Drug Location Concentration
µg L-1
Reference
Antibiotics
Ciprofloxacin
Korea 5.03
[169]
Ciprofloxacin Switzerland 31.9 [129]
Ciprofloxacin Spain 15 [170]
Ofloxacin Spain 22 [170]
Cefradine China 0.12 [171]
Ofloxacin China 0.38 [171]
Ciprofloxacin Portugal 3.67 [172]
Ofloxacin Portugal 7.30 [172]
Ofloxacin Spain 10.36 [173]
ciprofloxacin Spain 7.49 [173]
Ofloxacin France 0.7 [174]
Cefotaxime Spain 0.08 [173]
Cefotaxime Germany 0.49 [175]
Cefuroxime Germany 6.19 [175]
Ciprofloxacin Germany 1.57 [175]
Ofloxacin Spain 32 [175]
63
Ciprofloxacin Spain 12 [175]
Ciprofloxacin Spain 13.7 [176]
Ofloxacin Spain 4.75 [176]
Cefotaxime Spain 0.236 [176]
Analgesics
Diclofenac Korea 6.88 [169]
Diclofenac Spain 0.46 [170]
Ibuprofen Spain 0.81 [170]
Diclofenac Switzerland 0.83 [129]
Diclofenac Brazil 1.78 [177]
Diclofenac Jordan 6 [178]
Diclofenac Portugal 0.189 [172]
Ibuprofen Portugal 7.728 [172]
Ibuprofen Spain 10.3 [175]
Diclofenac Spain 1.01 [175]
Diclofenac USA 0.06 [179]
Ibuprofen USA 32.8 [179]
2.5 Removal of pharmaceuticals from water systems
The PhACs are present in aqueous environmental systems usually as in
dissolved phase. Therefore, removal of PhACs in treatment process key aspect is
the biodegradation in wastewater. The biodegradation can be performed in aerobic
condition for activated sludge treatment or anaerobically for sewage sludge
digestion. In one study, the degradation of diclofenac was observed in 10 days
however the biodegradation varied as effluents drug concentration varied [180-
181].
64
The behavior of pharmaceuticals on excretion commonly observed as
nonconjugated and conjugated polar metabolites. Hence, the conjugate metabolites
can easily escape the sewage treatment plant (STP). Consequently, the release of
active parent compound was also observed as reported in case of estardiol and some
analgesics. The biological degradation of micro-pollutants like PhACs observed to
be high degree with hydraulic residence time and gradually with time the sludge
become activated for treatment [182-183].
The processing of STP depends on the influent and effluent concentration,
accordingly then varied the construction, treatment technology, hydraulic retention
time and also with compatibility of season. For pharmaceuticals mostly efficient
removal reported in secondary treatment step. As reported the conventional
treatment plants are not successful for complete elimination of large number of
pharmaceutically active compounds [184-185].
In Finland it was reported that frequently used carbamazepine was not removed at
all in STP, whereas only 40% of metoprolol was removed [186].
Also, the anticancer drug tamoxifen (antiestrogen) was not eliminated. The
elimination rate of PhACs in STPs have different pattern for each and every drug.
Pharmaceutical compounds have the chemical miscellaneous nature, hence this
heterogeneity cause the inefficient remediation for all PhACs at once. The process
of remediation in STPs randomize due to various parameters like steps of treatment,
operation tools, temperature and weather conditions. The DFS removal was
observed with different rates from 17-69% [187-188].
Different advanced processes such as ozonation, chlorination, presipitation,
electroplating, electrodeposition, electro cogulation(EC), electroflotation (EF),
electrooxidation, biological treatment, ultraviolet irradiation, nanofiltration (NF),
reverse osmosis (RO), and activated carbon as secondary or in some cases as
tertiary treatments have been used for the removal of persistent PhACs. In case of
choloriantion some of PhACs by products complexed with cholorine and the new
byproducts are more toxic or potential environmental hazard. Ozonation is very
65
efficient for antibiotics remediation but it also develop a concern that biologically
active by-products formation, which are also increasing bacterial resistance issues
in environment. Fouling is observed by the buildup of precipitates or biomass on
NF/RO membranes. For UV remediation high energy beam radiations are required
to disinfect the wastewater [72, 189-191].
A promising option for contaminant remediation is hydrochar [192-193] and
conversion of biomass into char like material is centre of attention since last decade.
Thermochemical transformation of biomass is usually carried out at elevated
temperatures of around 180-250ºC along with autogenous pressure condition. Coal
like product is formed as a result of aromatization and polymerization of biomass
[194].
The earlier investigations reveal the process of HTC is process for degradation of
biomass. The hemicellulose of biomass completely degraded around 200ºC, where
as lignin degraded at some extent from 200-260ºC, both hemicelluloses and lignin
degradation in short time are observed under hydrothermal conditions. The HTC
material is reported to free of loose dirt and structural ash as well. The HTC material
applied for the treatment of agricultural soil, treatment shows efficient removal of
heavy metals [79, 195].
Ying Yao examined the adsorption experiments for methylene blue. Findings from
his work indicate that engineered biochar, prepared from two low-cost materials
(clay and biochar), is a valuable adsorbent for removing contaminants from
aqueous solutions.
The water accessibility in Pakistan once was self sufficient country, although day
by day it becomes insufficient country to complete consuming demand of people.
It was reported elsewhere that water shortage observed in water availability from
1.299 m3 per capita in the 1996-97. This scarcity of water will increase up to 700
m3 by 2025. Hence, to struggle against the shortage of water the prior step is must
to keep water quality uptight from contaminations. In Pakistan, domestic
wastewater, hospital wastewater and industrial wastewater are directly discharged
66
into sewer system. The large amount of wastewater in Pakistan is drop down in to
water streams (rivers and sea) without any treatment. Only two cities have
biological treatment plants (BTPs) are Karachi and Islamabad, even these BTPs are
only capable to treat small portion of wastewater.
2.6 Summing up literature review
Close observation of literature related to analytical methods and removal of
PhACs reveals that there is growing need for analytical methods that could detect
low levels of parent compounds and their biodegradation products.
Chromatographic methods are of choice always for such multielement samples. It
is reported elsewhere the multi-residual method for PhACs is well accountable with
sample preparation like SPE.
Besides, screening of pharmaceuticals there is need to develop cheaper
technologies for cleaning of wastewater contaminated with pharmaceuticals.
Solid-phase extraction is one the approaches to be studied for such applications.
lso been reported for preconcentration and
removal of pharmaceuticals but in best of our knowledge there is no or very limited
reports are available on use of natural adsorbents for treatment of hospital
wastewater. Here, we will also explore the natural adsorbent which may serve as
cheaper material to clean the water systems from pharmaceuticals.
The targeted study was to investigate the presence of pharmaceutical compounds
in hospital wastewater. In this relationship, it was observed that antibiotics are
-lactam group
(cephalosporin and penicillin), quinolones along with analgesic anti-inflamentry
drugs are widely prescribed in hospitals. These persistent pharmaceuticals in
environment need to be removed from the wastewater prior its disposal to the fresh
water bodies. Consequently, some environmental friendly and economical
convenient removal methodologies will also be developed in this study. The
selection of pharmaceutical compounds was based on the occurrence, persistency
and need to develop analytical methods and removal strategies.
67
CHAPTER 3
RESEARCH METHODOLOGY
3.1 Research work plan
Following is the work plan of current study:
about
frequently prescribed pharmaceutical drugs to patients. The layout of
questionnaire is given below in Table 3.1.
From data collected, the following drugs were selected for screening:
fluoroquinolones (ofloxacin and ciprofloxacin), cephalosporins (cefradine,
cefuroxime and cefotaxim) and analgesics (Ibuprofen and Diclofenac).
Screened data of selected pharmaceuticals in hospital wastewater. This aim was
achieved by the use of reported separation techniques on (LC/MS), as needed
new methods were developed to identify antibiotics and analgesic classes of
compounds too.
Hospital wastewater samples collection were made at influent point. The
sampling sites are shown in Fig.3.1.
Removal of selected and persistent pharmaceutical compounds by screened data
on natural sorbent was done.
Treated sawdust used for removal of ofloxacin.
Treated peanut shells utilized for removal of ciprofloxacin.
Removal/Preconcentration efficacy of adsorbents was explored for selected
pharmaceuticals in hospital wastewater.
The techniques/instrumentation used for analytical study are listed below:
68
LC/MS
GC/MS
HPLC (UV/Visible and Diode array)
Simultaneous removals of ibuprofen and diclofenac sodium were done on
hydrothermal caboneous sorbent (Ziziphus mauritiana L.).
Table 3.1 Survey form of healthcare facilities for pharmaceuticals utilization
General Information of Healthcare facility
Name of Healthcare Facility
Address
Governance and Management authority
General prescription information
Patient Type of Facility Prescription
Detailed Information about hospitalized patients presc
ription
Category of Drug Generic name Dose prescript ion Duration of treatment Number of patient
Antibiotics
Analgesics
Lipid regulators
69
Antidepressant
Beta blockers
Diuretic
Anti epileptics
70
Figure 3.1 Map indicating the sample collection points of hospital wastewater
3.2 Chemicals and solvents
All solvents/reagents used for the experiments and preparations of solutions were
of HPLC and GC grades. Ofloxacin (OFL) and ciprofloxacin (CPLX) were bought from
MP Biomedicals, Inc., France. Hydrochloric acid (HCl, 37%), sulphuric acid (H2SO4,
99.9%), sodium hydroxide (NaOH, 98%) were purchased from Merck, Germany.
Methanol, acetonitrile, acetic acid, acetone and dichloromethane were purchased from
Fisher Scientific, UK. Sodium nitrite (NaNO2), potassium carbonate (K2CO3), magnesium
sulphate (MgSO4), Ibuprofen (IBP), diclofenac sodium (DFS), cephradine (CFD),
cefuroxime (CFX) and cefotaxime (CFT) were acquired from Sigma-Aldrich, Finland.
71
The deionized water purified by a Millipore Milli-Q Plus water purification system (Elga
model classic UVF, UK) was used to prepare aqueous solutions. Oasis® HLB cartridges
60 mg 3 mL-1 were purchased from SUPELCO Bellenfonte, USA. 0.45-µm cellulose
acetate membrane filter (Micropore) was purchased from Sigma-Aldrich.
3.3 Instrumentation
The analytical techniques and operational equipments used during this
study are given in Table 3.2.
Table 3.2 List of Instruments and operational equipments
1 The Ionlab pH 720(Germany) with glass electrode and internal
reference electrode was used to study pH
2 JEOL, JEM, 1200EX, Tokyo, Japan, .instrument was utilized to
perform scanning electron microscopy (SEM) with JEOL, JEM,
1200EX, Tokyo, Japan.
3 A Gallenkamp thermostat automatic mechanical shaker product
number BKS 305-101, UK was used for batch adsorption
experiments at desired temperatures
4 Automated SPE system SUPELCO VISIPREP was utilized to
perform SPE studies
5 A Hitachi 6010 liquid chromatograph fitted with a Hitachi L-4200
variable wavelength UV-Vis detector, a Rheodyne 7125 injector,
and a Hibar C-18, 250 mm x 4.6 mm i.d. column (Merck,
Germany) were used throughout the experimental. The CSW32
software (Data Apex) was used for data acquisition and integration
72
6 GC/MS Agilent 6900 connected to a mass spectrometer Agilent
5975 (Agilent technologies, USA)
7 LC-MS (LCQ Advantage Max, Surveyor with quadruple and ion-
trap system by Thermo Finnigan, California, USA) comprising a
Surveyor MS Pump and an autosampler
8 Milli-pore quality water was acquired from Milli-Q system
(ELGA Model CLASSIC UVF, UK)
9 Thermo Scientific FT-IR iS10 model spectrophotometer (USA) in
ATR mode
10 UV/Vis spectra were recorded on a Shimadzu UV-1601
11 Vortex Mixer V8, Nade, Zhejiang, China (Mainland)
12 The QUADRASORB-SI, Boynton Beach, Florida (USA)
3.4 LC/MS screening of ofloxacin, ciprofloxacin and diclofenac sodium
from hospital wastewater in Hyderabad, Sindh (Pakistan)
3.4.1 Sampling
73
Hospital wastewater samples were collected from outfall drain of five local
hospitals of Hyderabad, Pakistan.
Picture 3.1 Collection of hospital wastewater influent on cite (a &b) and sample
vaccum filtration in laboratry (c)
3.4.2 Collection of samples
Hospital wastewater influent was collected in Pyrex borosilicate amber glass
bottles. To ensure negligible experimental error all the glass wares were
thoroughly cleaned, oven dried at 320 °C for decontamination about 8 h and
finally rinsed with Millipore water before use. The collected samples were
transported to the laboratory in a portable icebox for immediate processing their
extraction was completed within 48 h. In order to keep away from samples
a)
74
spoilage and PhACs contaminants dilution or degradation a timely extraction
was carried on priority bases.
3.4.3 Sample pre-treatment of hospital wastewater
The filtration of samples was performed by using a cellulose acetate membrane
filter (micropore) of 0.45 µm pore size. The solid phase extraction cartridges
were used to pre-concentrate samples. The automated SPE system SUPELCO
VISIPREP was used to perform SPE.
3.4.4 Solid-phase extraction
Solid-phase extraction was performed on Visiprep Solid-Phase extraction
system fitted with mini vacuum pump (Supleco, Bellefonte, PA, USA) utilizing
Oasis® HLB cartridges 60 mg 3 mL-1 (Waters, Milford, USA) used reported
method for DFS [196] and for quinolone, CIP and OFL both were
preconcentrated by method reported elsewhere [197].
3.4.5 Instrumentation
Thermo finnigan LCQ advantage max LC/MS/MS ion trap spectrometer
coupled with SurveyorPlus photodiode detector and degasser, San Jose,
California, USA the autosampler operated at 20 µL sample volume for
identification of the compounds [196-197]. All the data was processed by using
the Xcalibur software.
75
3.5 LC/UV and LC/MS determination of cefradine, cefuroxime, and
cefotaxime
3.5.1 Sample collection and preparation
Hospital wastewater samples were collected from the out drain of Hospital of
Liaqat University of Medical and Health Sciences, Jamshoro, Pakistan in the month
of May 2011. Samples were filtered through 0.45 m filter paper and a 200 mL
aliquot was reduced to 10 mL using a rotary evaporator with vacuum pump V-700
(R-210 BUCHI) at reduced pressure keeping the temperature at 50 ºC.
3.5.2 Solid phase extraction
Visiprep Solid-Phase extraction system fitted with mini vacuum pump (Supleco,
Bellefonte, PA, USA) was employed for clean-up. Oasis® HLB cartridges 60 mg
3 mL-1 (Waters, Milford, USA) and C-18 50 mg 1mL-1 (Supelco, Bellefonte, PA,
USA) were used.
3.5.3 Instrumentation
A Hitachi 6010 liquid chromatograph fitted with a Hitachi L-4200 variable wavelength
UV-Vis detector, a Rheodyne 7125 injector, and a Hibar C-18, 250 mm x 4.6 mm i.d.
column (Merck, Germany) were used throughout the study. The CSW32 software (Data
Apex) was used for data acquisition and integration.
An LC-MS (LCQ Advantage Max, Surveyor with quadruple and ion-trap system by
Thermo Finnigan, California, USA) comprising a Surveyor MS Pump and an autosampler
with 20 µL sample volume was used for identification of the compounds.
All the data was processed using the Xcalibur software.
3.5.4 LC and LC/MS conditions
Separation was carried out with a mobile phase composition of methanol and 0.05%
formic acid (55:45) at a flow rate of 1.0 mL min-1. The sample injection volume
was 20 L, while UV detection was carried out at 260 nm.
76
LC/MS was operated on similar conditions as mentioned for LC-UV. MS was
operated in ESI (positive ion) mode; needle voltage of 4.5 kV, probe temperature
of 200oC, cone voltage of -29.6V, sheath gas flow rate of 53 arbitrary units, and
auxiliary gas flow rate of 43.6 arbitrary units. Samples were run in SIM mode with
three selected ions; 349.82, 455.50 and 447.8 for cefradine, cefatoxime and
cefuroxime, respectively.
3.5.5 Synthetic and hospital wastewater samples
The optimized method for preconcentration and chromatographic separation was
employed to determine three cephalosporins in synthetic wastewater and hospital
wastewater. Composition of synthetic wastewater is given in table 3.3 [198]. Since,
hospital wastewater contains many unknown compounds; the samples were run
using same liquid chromatographic procedure with mass spectrometric detection.
Also, spiked synthetic wastewater was run parallel to hospital wastewater to check
the performance of assay procedure.
Table 3.3 Composition of synthetic wastewaters used for recovery studies of
various drugs
Chemical
Compounds
Conc.
(mg L-1)
Food
Ingredients
Conc.
(mg L-1)
Trace
metals
Conc.
(mg L-1)
Urea
91.74
Starch
122
Cr(NO
) .9H O
0.770
NH4Cl
Na-acetate
Na-acetate.3H2O
Peptone
MgHPO4
KH2PO4
FeSO4.7H2O
12.75 79.37
131.64
17.41 29.02
23.4
5.80
Milk powder
Yeast
Soy oil
116.19
52.24
29.02
3 3 2
CuCl2.2H2O
MnSO4.H2O
NiSO4.6H2O
PbCl2
ZnCl2
0.536
0.108
0.336
0.100
0.208
77
3.6 Determination of ibuprofen drug in aqueous environmental samples
by gas chromatography mass spectrometry without derivatisation
3.6.1 Preparation of solutions and samples
Water samples were collected from local hospitals of Hyderabad, Pakistan. After
immediate transfer to laboratory, samples were adjusted to pH 2.5 with HCl (37%)
and were vacuum filtered using 0.45 µm filter paper. Samples collection, filtration
and enrichment were completed on the same day to avoid any loss of
IBP in analysis
3.6.2 Solid-phase extraction
The solid-phase extraction was performed on Visiprep Solid-Phase extraction
system fitted with mini vacuum pump (Supleco, Bellefonte, PA, USA). The Oasis®
HLB cartridges 60 mg 3 mL-1 (Waters, Milford, USA). The method reported by for
extraction of IBP from aqueous samples with little modification was used. Briefly,
cartridges were sequentially conditioned prior to sample loading, with methanol (5
mL) and ultrapure water adjusted to pH 2.5 (5 mL) at flow rate of 1 mL min-1.
Samples were also prepared with various IBP concentrations in 100 mL of synthetic
wastewater, river and hospital wastewater for recovery studies. Samples were
loaded onto SPE cartridges under vacuum and maintained with a constant flow rate
of 10 mL min1. The cartridges were then dried under a gentle flow of nitrogen gas.
Ibuprofen was eluted from the SPE cartridges with two fractions of 2× 4 mL of
methanol at flow rate of 1 mL -1and then evaporated completely under mild nitrogen
stream. The volume was made up to 1mL with dichlorometane prior to GC/MS
analysis. By this process, samples were preconcentrated 100 times to its original
concentration
3.6.3 Standard solution
The IBP standard solutions of 10 µg mL-1 were prepared in methanol and
dichloromethane. For further studies and calibration plot stock solutions of IBP
78
(1000 µg ml-1) were prepared in dichloromethane and successive dilutions were
done.
3.6.4 Synthetic wastewater
Synthetic wastewater was prepared by mixing various nutrients, minerals, etc.
reported composition was employed (Table 3.3) [199] .
3.6.5 Chromatographic conditions for determination of ibuprofen
The Agilent 6890 gas chromatograph with 7683 autoinjector, and also mass
selective detector 5973 MSD with turbo pump were used for ibuprofen
determination. Chromatographic determination was performed with a HP5 MS
capillary column (30m, 0.25mm I.D., 0.25µm thickness). To achieve the
determination programming of temperature of column was initially at 100 ºC, with
1 min hold, the temperature degree rise from 100 to 300 ºC with the ramp rate of
oven temperature was 10ºC per minute. The injector port was operated at 300 ºC,
helium as carrier gas was flowed at 7.7mL min-1. Mass detector was operated at
350ºC. The splitless mode was utilized to run 1 µL samples aliquot with 1 minute
hold. The ionization source for IBP determination was used as electron impact (EI)
70 eV. Full-scan mass spectra of the derivatives were observed in the range of m/z
50-600. Selected ion monitoring (SIM) was employed for the quantitative analyses
m/z of
206, 163, 161,119 and 91 that are characteristic fragment ions of IBP.
79
3.7 Removal of ofloxacin onto sawdust (Erythrophleum suaveolens
L.)
3.7.1 Preparation of sorbent
Sawdust sieved (600 µm) size was washed with water, 0.1N HCL and 0.1N NaOH
to clean the surface residual and bring in neutral, acidic and basic surface activities.
A 4 g of SD was weighed out in conical flask and 100 mL of 0.1N HCl were added
and placed in shaker for 30 minutes at 120 rpm. After shaking SD was filtered out
and washed with deionized water till filtrates pH became neutral. Treated SD was
oven dried overnight at 80°C and kept in closed bottle for further use. Same steps
were repeated using 0.1 N NaOH for basic treatment.
3.7.2 Sample collection
Hospital wastewater samples were collected from outfall drain of four local
hospitals of Hyderabad, Pakistan. Amber glass bottles were used to collect samples
from each site. After immediate transfer to laboratory, samples were vacuum
filtered using 0.45 µm filter paper. Samples collection, filtration and enrichment
were completed on the same day to avoid any loss of ofloxcin in analysis.
3.7.3 Synthetic wastewater
Synthetic wastewater was prepared by mixing various nutrients, minerals, etc.
using reported composition (Table 3.3)[198].
3.7.4 Solid-phase extraction
The solid-phase extraction was performed on Visiprep Solid-Phase extraction
system fitted with mini vacuum pump (Supleco, Bellefonte, PA, USA) utilizing
Oasis® HLB cartridges 60 mg 3 mL-1 (Waters, Milford, USA) using reported
method.
3.7.5 Mathematical and statistical procedure
Multi-variant sorption optimization was performed using Draper-Lin small
composite design. The design contained eighteen batch experiments; each
80
experiment was performed at the different level of independent variables.
Statgraphics Centurion (XVI) from Statpoint Technologies, Inc, USA was used for
all calculations of CCD.
3.7.6 Sorption equilibrium of ofloxacin onto sawdust (SD)
The equilibrium sorption of the ofloxacin (OFL) was carried out by contacting
0.5gm of HCl treated SD with 0.02 L volume of different concentrations from 1 mg
L-1 100 mg L-1, agitated for 150 minutes on the rotary shaker. The residual
concentration of OFL was analyzed by HPLC. The % removal was calculated by
Eq. 3.1.
(Eq. 3.1)
Where Co is the initial concentration and Ce is equilibrium concentration (µg L-1).
All experiments were carried out in triplicate in order to minimize the error. The
data was fitted into the following isotherms Eq.1.1-3: Langmuir, Freundlich and
Dubinin-Raduskevich (D-R).
3.7.6.1 Langmuir isotherm
This describes the formation of a monolayer sorbate on the outer surface of the
sorbent, after monolayer formation no further sorption take place. The Langmuir
model supports uniform energies of adsorption onto a surface of identical and
localized adsorption sites. The linear form of Langmuir equation [200] is given
Table (3.4)
3.7.6.2 Freundlich isotherm
Freundlich isotherm commonly used to describe the adsorption characteristic of
heterogenous surface. The linear form of Freundlich equation [201] is given in
(Table. 3.4).
3.7.6.3 Dubinin-Radushkevich isotherm
81
This isotherm is generally applied to express the adsorption system with a Gaussian
energy distribution. The linear form of equation [202] is given in (Table. 3.4) . In
equation is Polanyi potential and is equal to RT ln (1+1/Ce), T is
of adsorption per mole of the adsorbent when it is transferred from infinite distance
in the solution to the surface of the solid.
Table 3.4 Langmuir, Freundlich and D-R isotherm equations and parameters
Linear Equation Parameter
Langmuir Isotherm
Ce = equilibrium concentration of
sorbate (mg L-1)
qe = the amount of sorbate adsorbed
per gram of sorbent at equilibrium
(mg g-1)
qmax = maximum monolayer
coverage capacity (mg g-1)
KL = Langmuir isotherm constant
n = adsorption intensity Ce =
equilibrium concentration of
adsorbate (mg L-1) qe = amount of
sorbate adsorbed per amount of
sorbent at equilibrium
(mg g-1)
82
KD-R = Dubinin-Radushkevich isotherm
constant
mole of the adsorbent (mol2 k-1j-2)
= Polanyi
potential
3.7.7 Adsorption kinetics of ofloxacin onto SD
The kinetics of adsorption is directly related to competence of adsorption process.
The kinetics of OFL was studied from the time versus %removal plots. The rate
kinetics of OFL adsorption onto HCl treated SD was analyzed by using three kinetic
models namely pseudo first-order [203], pseudo second order [204]and Morris
Weber [205] to test the experimental data using equation 3.2, 3.3 and 3.4,
respectively.
(3.2)
(3.3)
(3.4)
qe and qt (mg g-1) are the adsorption capacities at equilibrium and at time t
respectively. k1 (min-1) is the rate constant of pseudo-first order adsorption, k is the
equilibrium rate constant for second order rate equation. Kd intra-particle diffusion
rate constant (mg g-1min0.5) and C intercept which gives the idea of thickness of the
boundary layer.
83
3.8. Removal of Ciprofloxacin (CIP) onto peanut shells (Arachis
hypogaea L.)
3.8.1 Preparation of sorbent
The peanut shells (PS) were collected from local market of Hyderabad. PS were
thoroughly washed with distilled water to remove dust particles and then dried in
oven at 80ºC for 24 h. PS was crushed and sieved 420 µm sized. A 4 g of PS was
weighed out in conical flask and 100 mL of 0.1N HCl were added and placed in
shaker for 30 minutes at 120 rpm at 25ºC. After shaking PS was filtered out and
washed with deionized water till filtrates pH became neutral. Treated PS was oven
dried overnight at 80°C and kept in closed bottle for further use.
3.8.2 Batch study for removal by response surface methodology
A batch sorption experiments were carried out on a water-bath, thermostate shaker
(Gallenkamp model BKS 305-101,Uk). Batch adsorption was held at 303 K for CIP
onto PS. At constant shaking speed the batch experiments were performed. Each
sorption experiment in conical flask contains 20 mL of aqueous sample hold known
concentration of sorbate and sorbent. The adsorption reaction flasks were agitated
at constant 100 rpm. Effects of various parameters were studied by Multi-variant
sorption optimization by using Draper-Lin small composite design. The design
contained eighteen batch experiments; each experiment was performed at the
different level of independent variables. All the trials were performed in triplicates
and the averages of corresponding recovery percents were treated as responses.
3.8.3 Adsorption isotherms of CIP onto treated PS
Batch adsorption experiments were conducted onto acid treated PS by using 25 mL
stoppered conical flask containing 20 mL of aqueous samples. Temperature kept
303 K, containing 0.135 gm of Acid treated PS adsorbent. Various initial
concentration of CIP was used at pH 8, from 5 mg L-1 to 100 mg L-1. All flasks
were shaken at 100 rpm using a thermostated shaker for 70 minutes. For alkali
treated PS 0.1 gm of adsorbent was agitated with aqueous sample at pH 9.5, rest
84
conditions kept same as used for acid treated PS adsorption studies. The
equilibrium of the adsorption process for the CIP removal by using acid and alkali
treated PS, both were evaluated by applying Langmuir isotherm, Freundlich
isotherm and Dubinin-Radushkevich isotherm equations (Table 3.4) for obtained
experimental data.
3.8.4 Adsorption kinetic studies of CIP onto treated PS
The kinetics of CIP adsorption was studied from the time versus percent removal
curves. The rate kinetics of CIP adsorption onto acid treated PS and alkali treated
PS was analyzed using the pseudo first-order, pseudo second order and Morris
Weber to test the experimental data using equation 3.2, 3.3 and 3.4, respectively.
3.8.5 Surface characterization
Acid treated peanut shell was better option for CIP removal, further its surface
morphology was examined by scanning electron micrographs (SEM). Sample was
hold and sputter for 2 min for coating with Au and analyzed with JEOL, JEM,
1200EX, Tokyo, Japan. Textural information of sorbent was collected by nitrogen
adsorption at 77K by Brunauer-Emmetl-Teller (BET) BJH model used (The
QUADRASORB-SI, Boynton Beach, Florida, USA).
N2 adsorption desorption at 77 K by Brunauer-Emmetl-Teller (BET) BJH model
used for specific surface area, the total pore volume and the mean pore diameter
were measured, using a QuantaSorbSI instrument (Quantachrome, USA). Prior to
the adsorption desorption measurements, sample was degassed at 200 °C in a N2
flow for 3 h to remove the moisture and other adsorbates.
3.8.6 Removal from synthetic wastewater and hospital wastewater
Prior to CIP batch sorption at optimized conditions synthetic wastewater (section
3.3) and all five hospital wastewater samples 50 mL of each sample was spiked,
vortex for 30 seconds and filtered with 0.45 µm filter paper. The filtrated samples
were run for batch sorption process for CIP removal onto acid treated PS with
design obtained optimum conditions.
85
3.9 Simultaneous removal of Ibuprofen and Diclofenac sodium onto
HTC of Ber (Ziziphus mauritiana L.)
3.9.1 Preparation of hydrothermal carbon from ber fruit
Dried fruit samples were taken from the store of NCEAC which were stored after
a study on varieties of Ziziphus Mauritiana L.Fruits. Sampling details are given in
a published paper [206].
86
The reaction was carried out in a nonstirred, 30 mL capacity Teflon-lined stainless
steel autoclave, which was put in an oven. A 6 g of Ziziphus Mauritiana L. fruit
pulp was dispersed in 15 mL of deionized water and 10.0 mg of citric acid was
added to ensure acidic carbonization conditions. The autoclave was sealed and
placed in oven at 200 °C for 20 h and then allowed to cool to room temperature
[207]. The products were filtered off, washed several times with distilled water and
absolute ethanol, and finally dried in an oven at 80 °C for 4 h. Yield of HTC was
calculated by following equation:
(Eq.3.5)
3.9.2 Surface characterization
Thermo Scientific FT-IR iS10 model spectrophotometer (USA) in ATR mode was
used to characterize surface functional groups. Scanning electron microscopic
(SEM) imaging was made by Raster electron microscopy, Leitz-AMR-1000,
Germany. N2 adsorption desorption experiment by using BJH method, specific
surface area the total pore volume and the mean pore diameter were measured using
a N2 adsorption desorption isotherm at liquid nitrogen temperature (77 K), using a
QuantaSorbSI instrument (Quantachrome, USA). Prior to the adsorption desorption
measurements, sample was degassed at 300 °C in a N2 flow for 3 h to remove the
moisture and other adsorbates.
3.9.3 Batch adsorption experiments
Batch mode experiments were carried out to study the adsorption capacities of the
hydrothermal carbonization of Z. Mauritiana L. (HTC-ZM) sorbent used in the
experiments in a thermostated shaker keeping the temperature constant at 303 K.
For parametric optimization, different amounts of HTC-ZM were agitated with
solution containing different concentrations of diclofenac sodium and ibuprofen at
pH values 2-8 for a period of 10-120 minutes at 120 rpm. The Draper-Lin
composite was used to evaluate the basic parameters affecting sorption process.
87
All the experiments were done in duplicate and random order to evaluate pure error
and minimize the effect of possible uncontrolled variables. The response function
coefficients were determined by regression using Statgraphics plus 5.1 computer
program.
3.9.4 Adsorption isotherm study of DFS and IBP
The adsorption isotherm data for DFS and IBP were analyzed by three
mathematical isotherm equations (Langmuir, Freundlich and
DubininRadushkevich) given in Table 3.4. Isotherms data was obtained by taking
250mg of sorbent and using 20 mL of various concentrations of drugs; DFS and
IBP at 303 K.
3.9.5 Adsorption kinetics of DFS and IBP
The kinetics of DFS and IBP sorption process onto HTC-ZM were analyzed in the
light of three well known kinetic equations: pseudo first-order, pseudo second
order and Morris Weber (section 3.7.7). Kinetics data was obtained by taking 250
mg of sorbent and 20 mL solutions of 10 µg mL -1 and 100 µg mL-1 of each drug;
DFS and IBP. Kinetics data was obtained up to 1140 minutes at various time
intervals.
3.9.6 Removal of DFS and IBP from Spiked synthetic wastewater
Synthetic wastewater was prepared by mixing various nutrients, minerals, etc.
using reported composition (Table 3.3)[198]. Prior to filtration 50 mL of sample
was spiked and vortexed for 30 seconds. Spiked synthetic wastewater and hospital
wastewater samples then run for batch sorption removal onto HTC-ZM.
88
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 LC/MS screening of pharmaceutical compounds from hospital wastewater in
Hyderabad, Sindh (Pakistan)
Results of targeted screening analysis of ciprofloxacin (CIP), ofloxacin
(OFL) and diclofenac sodium (DFS) from hospital wastewater samples are shown
89
in Table 4.1. These drugs were screened on LC/MS by reported methods [196197]
and found within the following ranges: OFL 17-53 µg L-1, CIP 70-164 µg L-
1 -1 -1 and DFS 78 - 118 µg L . All three drugs were found in µg L level in nearly all
samples. Our findings are similar to reported literature in other parts of the world.
Among variety of synthetic drugs the antibiotics and non steroidal
antiinflammatory drugs (NASIDs) are highly consumable drugs worldwide. Their
frequent use is one of the major causes towards availability of antibiotics and
NASIDs in water bodies [208-210]. Moreover frequent prescription to hospitalized
patients and their incomplete metabolism and poor degradation in environmental
waters leads to their higher concentrations in hospital wastewaters.
Table 4.1 Hospital wastewater screened samples by LC/MSMS for ciprofloxacin,
ofloxacin and diclofenac sodium
Drug Concentration (µg/L) (± S.D)
Hospital A Hospital B Hospital C Hospital D Hospital E
OFL 17( ± 0.14) 49( ± 0.9) 53( ± 0.12) 43( ± 0.7) 51( ± 0.9)
CIP 70( ± 0.21) 95( ± 0.4) 164( ± 0.72) 135( ± 0.3) 84( ± 0.1)
DFS 105( ± 1.2) 78( ± 1.8) 97( ± 2.5) 85( ± 1.6) 118( ± 2)
4.2 Determination of ibuprofen drug in aqueous environmental samples
by gas chromatography mass spectrometry without derivatisation
Note: Work of this part is based on published article with the following reference,
Qureshi etal., AM. J. Chromatogr (2014) Vol. 1 No. 1 pp. 45-54.
Ibuprofen is an acidic drug, soluble in polar solvents and remains in
protonated (neutral) form at acidic pH. IBP has high melting and boiling point to
be directly analyised by gas chromatography, however, its sublimation increases as
temperature increases. Thus, the vapors of sublimed IBP in injector ( 300 C)
90
are taken to capillary column and carried to mass detector via carrier gas.
Figure 4.1 (a & b) The IBP standard of 10µg mL-1 (a) ion chromatogram & mass
spectra (b) at full scan mode
Figure 4.1(b) shows the extracted ion chromatogram of IBP using m/z; 206, 163,
161, 119, and 91 as selected ions and full scan mass spectrum and fragmentation of
IBP in using electron impact (EI) ionization. For identification of IBP in unknown
samples, spectral match with NIST05 library was used whereas quantification and
optimization of IBP was carried in selected ion monitoring mode (SIM) using ions
206, 163, 161, 119, and 91 ions (Table 4.2). Multiple ions were used to enhance the
signal thereby the sensitivity of assay.
Table 4.2. IBP fragment ions with m/z
91
4.2.1 Method optimization
To optimize chromatographic separation of IBP different temperature programs
and modes of injection were tested. Chromatographic conditions; including mode
of injection (split or split less), injector temperature, volume of sample injection,
final temperature, flow rate of carrier gas and ramp rate of
oven were optimized.
4.2.1.1 Injection parameters
IBP standard was prepared in methanol in order to maintain the solvent match to
that eluted from SPE, initially. Standard prepared in methanol gave poor signal
even for 10 µg mL-1 of IBP (Figure 4.2); however, signal was significantly
increased when standard of same concentration of IBP was prepared in
206
163
161
119
91
92
dichloromethane. This may be due to hydrogen bonding or other polar interactions
of methanol with IBP which diminishes entropy, thereby sublimation rate.
Figure 4.2 (a & b) Response IBP standard prepared in dichloromethane (a) and
methanol (b)
4.2.1.2 Sample volume and injection mode
Using sample volume of 1µL (10 µg mL-1) of IBP was injected in split and splitless
mode at injector temperature 300C. No peak was observed using split mode
with split ratio of 1:10 even on injecting 3 µL of sample volumes. However, poor
signal was observed when same concentration of IBP was injected at split ratio of
1:50. Therefore, splitless mode was studied further. Volume of injection was
optimized from 0.2 to 2 µLs where 1 µL in splitless mode provided highest signal
along with better peak shape whereas on 0.2 µL no appreciable signal was observed
and at 2µL very large signal was produced with broad peak shown in Figure 4.3 (a
& b).
93
Figure 4.3 (a & b) Response IBP (10 µg mL-1) using 1 µL injection volume split mode
(1:50) (a) and splitless mode (b)
4.2.1.3 Temperature programming
Various initial (100 to 350C) and final (250 to 450C) temperatures with ramp rates
of 10 to 30C min-1 were tried to get the IBP peak with total run time of 10
minutes. Also, the peak intensity and shape were taken in to account in selection of
optimum temperature parameters. The suitable parameters were as 100C as
initial temperature hold for 1 minute and then increased temperature with rate of
10C min-1 and reached to 300 C and then held for four minutes.
4.2.1.4 Detector parameters
Throughout the study, MS detector temperature was set at 350C. MS was operated
at 50-600 amu in scan mode while in SIM mode targeted ions were 91,
119, 161,163, and 206 m/z these ions used for quantification/optimization of IBP.
94
4.2.2 Method validation
The LOD and LOQ for IBP were determined as the lowest absolute amount of
analyte detected with signal-to-noise ratios of at least 3:1 and 10:1, respectively,
with correct relative ion intensities and a retention time. LOD and LOQ for IBP
were found to be 0.08 µg mL-1 and 0.26 µg mL-1, respectively. Moreover, the
samples can be concentrated 100 times, therefore, the LOD and LOQ of 0.8 ng mL-
1 and 2.6 ng mL-1 may be assumed, respectively. The linearity of method was
determined by the calculation of the regression line using the method of least
squares with r2 = 0.994 with slope (a) 1.04 0.03 and intercept (b) -3.36 0.93,
the linear range of method was 0.8 to 70 µg mL-1 analyzed in triplicate in SIM mode
(91, 119, 161,163, and 206). The accuracy and precision were investigated at three
concentration levels (10, 20 and 50 µg mL-1) of IBP in the linear range with five
independent replicates on the same day and on three consecutive days (Table 4.3).
Table 4.3 Accuracy and precision data assay of IBP in Synthetic wastewater
Drug
Concentration
µg mL-1
Intra-Day
% RSD *(±SD)
Inter-Day**
% RSD(±SD)
%Recovery
Ibuprofen
10
1.42±0.06
2.68±1.2
80
20 4.01±2.08 9.54±3.41 85
50 1.5±1.04 2.09±1.47 114
Average from five replicate determination*, Average from three days determination**
4.2.3 Recovery of IBP
In order to establish the reliability of the reported method for extraction of IBP, recovery
experiments were carried out. Apparent recoveries, calculated as the ratio of the measured
concentration in calibrated levels to the spiked synthetic wastewater (expressed as
percentage). For recovery, known amounts of IBP added whose concentration after
95
preconcentration reached at 10, 20 and 50 µg mL-1 in 100 mL of synthetic wastewater; the
observed recoveries were 80%, 85% and 114% respectively.
All samples were free of co-eluting peaks at the retention time of IBP which provided little
chances of positive error in identification of IBP. The selectivity of the method was
adequate with minimal matrix effect in the samples.
The developed method can be achieved with 80 to 114 % recovery to detect the IBP in ng
mL-1 level after preconcentration without derivatization. This is the first report on the
quantification of IBP without derivatization using GC-MS which offer simple and rapid
alternative to lengthy derivatization based procedures, whereas current procedure do not
involve any additional step but sublimation of IBP in injector port form the basis of assay
procedure.
4.2.4 Real water samples
The real water samples were analyzed using developed procedure by spiking 100ng mL-1
and without spiking, the obtained results are shown in Table 4.4. The presence of IBP found
in ng mL-1 level in nearly all of the samples which is higher as compared to rest reported
methods, mainly due to inefficient waste treatment plants in locality.
Table 4.4. Determination of IBP in real water samples followed by SPE
Sample Conc.(ng mL-1) ± SD
Unspiked Spiked*
Hospital
wastewater sample A
52 ± 0.12 162 ± 0.07
sample B 80 ± 1.13 174 ± 1.02
sample C 55 ± 0.014 155 ± 0.212
sample D 58 ± 0.04 149 ± 0.02
*Each sample was spiked with 100 ng mL-1 prior to preconcentration step.
96
4.3 LC/UV and LC/MS determination of selected cephalosporins:
cefradine, cefuroxime and cefotaxime
Note: Work of this part is based on published article with the following reference,
Qureshi et al. Springerplus, (2013), 2(1), 575.
Separation of cefradine, cefuroxime and cefatoxime was initiated using RPHPLC
with formic acid in mobile phase as pH adjuster. Various parameters like; mobile phase
composition, concentration of formic acid, flow rate, detection wavelength and solvent for
sample were investigated. Organic modifier (methanol) was varied in the range 52-71 %
with neutral and acidified water (formic acid in the range of 0.05-0.1 % was used). Mobile
phase was found to induce pronounced effect on separation. Increase in methanol content
increased the retention times while increasing aqueous content eventually merged the three
components. Increase in retention time with increasing organic modifier may be due to
methanolysis of cephalosporins at higher methanol content. Also basic pH was not good at
resolving components due to ionization of compounds. Good separation in adequate time
97
was achieved with 55 % methanol and 45 % formic acid (0.05 %) modified aqueous phase.
Increase in strength of formic acid increased the retention while separation remained
unaffected.
Figure 4.4 (a ) Structures of three cephalosprins; cefradine, cefatoxime and
cefuroxime
Figure 4.4 (b) Separation of three cephalosprins; (1) cefradine (2.753), (2)
cefatoxime (3.740) and (3) cefuroxime (4.533) using 55%methanol+45%
98
formicAcid(0.05%) at flow rate of 1 mL min-1 and max 260 nm
Figure 4.3(a & b) shows the chromatogram obtained under optimized conditions,
all the three compounds are baseline separated within 5 minutes. The theoretical
plates were observed as; 9458 for cefradine, 6058 for cefuroxime and for
cefotaxime 6457, where as the resolution were observed for three peaks as 1.08,
2.76 and 1.09. The asymmetry for these three drugs was 1.4, 1.5 and 1.5 for
cefradine, cefuroxime and cefotaxime respectively.
4.3.1 Analytical figures of merit
Using optimized conditions linear calibration graph for cefradine range 5-20 g mL-1
(R2= 0.979), cefuroxime 0.5-15 g mL-1 (R2=0.998) and cefotaxime 1.0-20 g mL-1
(R2=0.999) were established. The intra-day (n=6) and inter-day precisions are shown in
Table 4.5.
Table 4.5 Intra-day and Inter-day precisions for cefradine, cefuroxime and cefotaxime
Cephalosporin
Concentration
µg mL-1
Intra-
Day
%
RSD
* InterDay**
% RSD
Cefradine 5 3.17 1.09
15 0.50 1.30
25 0.79 0.25
Cefuroxime
5
2.58
0.28
15 0.44 0.37
25 2.35 0.62
99
Cefotaxime
5
2.80
1.97
15 1.83 0.62
25 1.03 0.27
* Average from six replicate determinations, ** Average from five days determinations
4.3.2 Induced hydrolysis of cephalosporins
Cephalosporins undergo hydrolysis under various conditions like in the presence
of metals, acid/base or enzymes. However, the extent of hydrolysis for specific
cephalosporin varies significantly which depends upon the reaction conditions and
structure of the drug.
In this study, hydrolysis was induced using HCl (1M) and NaOH (1M) for acid and
base hydrolysis, respectively at elevated temperatures (70 C). Figure 4.5 shows the
chromatogram of each drug after acid hydrolysis. Cefradine (a) decomposed 21 %,
cefuroxime 100 % (b) and cefatoxime 92.8% (c) using induction time of 10
minutes. Moreover, cefradine was degraded into two more compounds; peak 1 A
and 3 A while peak 2 A is parent compound whereas cefuroxime showed three
additional peaks 1 B, 2 B and 3 B where as peak 4 B is parent compound and
cefatoxime showed two additional peaks 1 C and 3 C, peak 2 C is parent compound.
100
Figure 4.5 (a-b) Acid induced hydrolysis (1 M HCl) for 10 minutes at 70 ºC cefradine
(a), cefuroxime (b)
Figure 4.5 (c) Acid induced hydrolysis (1 M HCl) for 10 minutes at 70 ºC cefatoxime
Base hydrolysis completely decomposed all the drug compounds (98-100%
degradation) as only one distorted peak was observed for cefradine and cefatoxime
and many small peaks for cefuroxime with no peak at their corresponding retention
times. The data suggests that base hydrolysis is fast and induce more rigorous
conditions for selected cephalosporins.
101
4.3.3 Room temperature degradation of aqueous solutions
Figure 4.3.3 shows the degradation of three drugs in aqueous solutions at room
temperature. 20% cefradine, 30% cefouroxime and cefatoxime degraded during 30
h. The degradation was found faster at initial five hours then slowed down and
remained constant after 10 hour to 28 hours.
4.3.4 Sample preconcentration and clean-up
Solid phase extraction is common technique to clean sample and preconcentrate
pharmaceutical compounds. Various sorption materials; C-18, ENV+, HLB and
other are reported whereas hydrophilic-lipophilic polymeric phases are reported
-lactams [211] and widely accepted because of their capability to
preconcentrate/clean-up a wide range of compounds. However, most of the
methods have reported recoveries at higher concentrations (>/= 1µg mL-1) while in
real wastewaters lower µg or ng mL-1 ranges are usually observed. So, solutions
containing low concentrations of three cephalosporins were prepared and 200 mL
of each antibiotic was loaded onto HLB cartridges for preconcentration and eluted
with various solvents as shown in Table 4.6 (a) to achieve the better recoveries,
other material (C-18) as single phase or in combination was also tried.
Figure 4.6 Response at room temperature aqueous solution stability of cefradine,
cefuroxime and cefatoxime
20 5
102
Adsorption of cephalosporins onto SPE materials and then recovery studies were
initially carried out for single drug, and then all three drugs were loaded in mixture.
The recoveries of three cephalosporins in low ng mL-1 range (10, 10 and 50 ng mL-
1 for cefuroxime, cefatoxime and cefradine, respectively) using reversephase and
HLB type cartridges were calculated. Cefatoxime and cefuroxime showed recovery
of 79 % and 47% respectively when run as single compound while cefradine 90%
recovery on C-18 with elution solvent using 55% MeOH and 45% aqueous formic
acid. When all three drugs are simultaneously loaded onto sorbent materials and
eluted with methanol and acetone or mixtures from HLB and MeOH + acidified
water from C-18, the recovery varied appreciably; cefatoxime and cefuroxime
showed even better recoveries using HLB cartridges and acetone as elution solvent
as compared to methanol.
Table 4.6 (a) Recoveries of cefuroxime, cefatoxime and cefradine using SPE at various
concentrations and with different elution solvents
Drug Conc. Final Volume
(ng mL-1)
Adsorbent Elution solvent Recovery
(%)
Drugs optimized
Cefuroxime
individually
10 2 HLB 80% MeOH 5
Cefatoxime 10 2 HLB 80% MeOH 24
Cefatoxime 10 2 HLB 100% MeOH 79
Cefuroxime 10 2 HLB 100% MeOH 47
Cefradine 20 C-18 55:45 MeOH:0.05% formic
acid 90
Recovery using
Cefatoxime
mixture of drugs
50
2 HLB 100% MeOH 26
Cefatoxime 50 2 HLB 2 mL MeOH with 8 mL
acetone
53
Cefatoxime 50 2 HLB 8 mL acetone 85
Cefuroxime 50 2 HLB 100% MeOH 29
Cefuroxime 50 2 HLB 8 mL acetne 73
Cefuroxime 50 2 HLB 8 mL acetone 77
Cefradine 50 2 HLB 100% MeOH 00
103
Cefradine 50 2 C-18 55:45 MeOH:0.05% formic
acid
78
Recovery with HLB and C-18 in
Cefatoxime 50
series with mixt
2
ure of drugs
Mixed
mode 8 mL acetone 104
Cefuroxime 50 2 Mixed
mode
8 mL acetone 44
Cefradine 100 2 Mixed
mode
55:45 MeOH:0.05% formic
acid
55
Recoveries at 1µg mL-1 concentr
Cefatoxime
ations
2 Mixed
mode
8 mL acetone 95
Cefurxime 2 Mixed
mode
8 mL acetone 83
Cefradine 2 Mixed
mode
55:45 MeOH:0.05% formic
acid
80
However, cefradine better responded onto C-18 with acidified methanol. Since,
same sorbent material did not show good response to all three drugs, both the
materials were connected in series; HLB followed by C-18 and 200 mL containing
50-1100 ng mL-1 of drugs was passed through cartridges, air dried and then drugs
were eluted separately using acetone for HLB and acidified methanol for C-18.
Both eluates were pooled; solvents were evaporated under nitrogen stream and then
volume was made up to 4 mL with mobile phase and injected onto HPLC system
for recovery studies. Cefatoxime showed very good recovery but cefuroxime and
cefradine proved to be poorly recovered. The reason may be low concentrations of
drugs which renders the favorable interactions of molecules for good adsorption
hence results in losses during recovery studies. On the other hand, recoveries at 1µg
mL-1 were higher and acceptable.
104
Table 4.6 (b) Stability of drugs at various temperatures
30 99.86 97.00 94.74 40 92.21 93.87 90.24
50 90.98 91.65 90.19
60 83.79 85.34 69.55
70 80.72 82.72 66.52
Evaporation of solvent is one of the techniques which can be used to enrich the low
concentration or bringing concentrations of trace compounds in the ranges suitable
for SPE. However, evaporation of water from samples by boiling-off may degrade
cephalosporins so possible degradation of three drugs was studied at various
temperatures are shown in Table 4.6 (b). At 50 0C, all the drugs were fairly stable
and this temperature was kept constant and pressure was reduced enough to remove
the water by evaporation using rotary evaporator. Table 4.6 (c) shows the recovery
of each drug after evaporative preconcentration and recoveries using evaporative
preconcentration after clean up on HLB and C-18 materials followed by elution
using acetone and methanol/formic acid at concentration of
0.05 µg mL-1 cefuroxime and cefotaxime, 0.1 µg mL-1 for cefradine.
Table4.6 (c). Sample preconcentration using evaporation
Drug % Recovery Sample
volume (mL)
Preconcentrated
volume (mL)
Cefatoxime 79 200 10
Cefuroxime 76 200 10
Cefradine 72 200 10
Standard solutions are prepared from pure drugs therefore do not require any clean-
up to run on instrument however, real samples require such treatments. Therefore,
105
wastewater samples were preconcentrated and cleaned-up by evaporation followed
by solid phase extraction using optimized extraction protocol.
4.3.5 Synthetic and hospital wastewater samples
The optimized method for preconcentration and chromatographic separation was
employed to determine three cephalosporins in synthetic wastewater and hospital
wastewater. Composition of synthetic wastewater is given in Table 3.3. Since,
hospital wastewater contains many unknown compounds; the samples were run
using same liquid chromatographic procedure with mass spectrometric detection.
Also, spiked synthetic wastewater was run parallel to hospital wastewater to check
the performance of assay procedure.
LC/MS procedure is given in section 3.5.2. Presence of each compound was
confirmed by matching retention time with that of standard and molecular ion peak.
Cefradine molecular ion peak of 349.82 at tR 3.24 minutes, cefotaxime 455.50 at tR
3.53 minutes and cefuroxime 447.8 (M+Na) at tR 3.9 minutes were used for
confirmation. Good recoveries (77.8 112.5 %) were observed for spiked synthetic
wastewater containing of 50 µg L-1 (cefuroxime and cefotaxime) and for cefradine
(100 µg L-1) but none of the drug was detected in hospital wastewater (Hyderabad,
Sindh). The data is consistent with previous reports and may be explained because
of degradation of drug compounds by complex matrix effects, sunlight and possibly
adsorption on soil. However, one recent report has demonstrated the identification
of cephalosporin in wastewaters.
106
4.4 Ofloxaic removal optimization onto treated sawdust (Erythrophleum
suaveolens L.) By response surface methodology (RSM)
Note: work of this part is based on published article with the following reference,
Qureshi et al. Desalin Water Treat, (2016), 57(1), 221-229.
In this study sawdust (SD) was treated with water, HCl and NaOH and
removal of ofloxacin (OFL) was optimized using RSM. Mathematical modeling
successfully reduces the number of experiments for optimization process with
reliable results.
4.4.1 Optimization of ofloxacin removal by sawdust
Adsorption experiment for OFL was carried out by batch adsorption at 35ºC onto
treated SD. To conduct adsorption CCD approach was applied, the variables were
choose as pH values 2-9, contact time from 10-180 minutes at constant shaker speed
at 120 rpm, code levels of design are shown in Table 4.7. The initial and final OFL
concentrations responses were analyzed on RP-HPLC. The % removal was
calculated by Eq. 3.1.
107
Eq. 3.1
Here Cº and Ce are denoted for initial and equilibrium concentration (µg L-1) of
analyte respectively.
Table 4.7 Variables code levels of OFL removal by CCD
Independent variable Code levels
-1 0 +1
pH, A 2 5.5 9
Amount (mg), B 10 30 50
Concentration (mg L-1), C 10 55 100
Time (min.), D 10 95 180
The experiments were run in triplicate for OFL adsorption on treated sawdust. Till
date the reported removals for OFL were achieved by uni-variant sorption
optimization. The main disadvantage of uni-variant sorption optimization is the
effect of interaction terms cannot be recorded. Response surface methodology
(RSM) facilitates to study variable parameters simultaneously, and the interaction
effect of variables can be evaluated.
On the premise of data to fit in polynomial equation the RSM provides support with
statistical and mathematical experimental model. The RSM approach is well
applied when the more than one variable are affecting the experimental data. This
multivariant variables perspective helps to optimize the different levels of variables
simultaneously.
RSM have been successfully employed in various fields of analytical chemistry to
minimize the number experiments. In this study RSM model with minimum
number of experiments were designed and validated by fitting computational
values with experimental values. Following section details the discussion on
optimization of sorption process through this model. In RSM the experimental
errors are assumed to be random. The most commonly use RSM design is central
108
composite design (CCD), it is a quadratic fit model. CCD contain information from
correctly schemed factorial experiment and also very useful in sequential
investigation.
4.4.2 Statistical analysis and model validation
Draper-Lin composite design was used to correlate observed and predicted %
removal of ofloxacin from the aqueous solution. Both experimental and predicted
values for the design of 18 experimental runs are summarized in Table 4.8.
Corelation coefficient of 0.999 was observed for experimental and predicted
removal for all three treated sorbents. The validity of linear equation for proposed
model was tested by plotting a residual plot. The difference between the expected
data values and obtained data values of dependent variables are called residuals.
Basically, residuals are the sum of deviation of obtained data from the regression
line. The residual plot in which horizontal axis is the independent variables data
points, whereas on the vertical axis the residuals. The residual plots of all three
sorbents (Fig. 4.7. a-c) show a fairly random pattern with scattered values around
the axis. Random dispersion of values around the horizontal axis validates the
fitness of linear regression model.
Table 4.8Experimental observed % sorption and predicted % sorption of ofloxacin
HCl Treated sawdust NaOH treated sawdust Water treated sawdust
Observed Fitted Observed Fitted Observed Fitted
Exp.
set %sorption ± SD %sorption %sorption ± SD %sorption %sorption ± SD %sorption
1 59.67 ± 1.2 59.82 63.01 ± 1.5 63.02 50.1 ± 0.18 50.01
2 29 ± 2.5 28.89 21.47 ± 2.6 21.56 32.1 ± 3.2 32.04
3 66.7 ± 0.18 67.13 18.13 ± 1.2 17.77 46.14 ± 0.43 46.38
4 61.5 ± 3.2 61.23 37.87 ± 5.2 37.81 55.12 ± 5.2 55.42
5 20 ± 0.43 19.89 44.24 ± 0.64 44.33 36.62 ± 3.5 36.56
6 32.95 ± 5.2 33.10 30.32 ± 0.82 30.34 36.78 ± 2.2 36.69
7 64.85 ± 3.5 64.88 69.71 ± 0.2 69.61 56.03 ± 5.2 56.14
8 50.7 ± 2.2 50.85 49.06 ± 0.61 49.07 49.07 ± 1.2 48.98
109
9 61.85 ± 5.2 61.23 37.82 ± 5.6 37.81 55.16 ± 1.42 55.42
10 77.84 ± 1.2 77.99 38.44 ± 3.2 38.45 90.06 ± 2.6 89.97
11 45.82 ± 1.42 45.85 70.39 ± 3.41 70.30 27.23 ± 1.2 27.34
12 66.77 ± 0.18 66.92 56.86 ± 0.81 56.87 39.02 ± 5.2 38.93
13 47.16 ± 3.3 47.31 41.84 ± 0.4 41.86 39.67 ± 0.64 39.58
14 95 ± 0.29 94.89 59.01 ± 0.2 59.10 25.806 ± 0.82 25.75
15 71.23 ± 3.2 71.09 29.11 ± 1.1 29.49 48.56 ± 0.2 48.14
16 62.12 ± 0.25 62.01 51.53 ± 4.2 51.62 34.42 ± 0.61 34.36
17 37.89 ± 5.3 37.92 64.45 ± 1.5 64.36 44.1 ± 0.2 44.21
18 72.85 ± 0.21 72.88 19.32 ± 0.25 19.22 7.97 ± 1.2 8.08
The residual plot in which horizontal axis is the independent variables data points,
whereas on the vertical axis the residuals. The residual plots of all three sorbents
(Figure 4.7 a-c) show a fairly random pattern with scattered values around the axis.
Random dispersion of values around the horizontal axis validates the fitness of
linear regression model.
110
Figure 4.7 (a-c) Residual plot for removal of ofloxacin onto treated sawdust (a)
HCl, NaOH (b) and water (c)
111
0 20 40 60 80 100 120
Standardized effect
Figure 4.8 (a-c) Shows the effect of pH, concentration, amount of sorbent and
contact time on the removal of ofloxacin
The acquired experimental data was fitted by different polynomial equations using
by Statgraphics software. The significance of all calculated effects were
test. The t values found were 2.76, 1.77 and 1.98 for SD HCl, SD NaOH and SD
water, respectively. The acceptable criterion for the experimental design is
considered valid when, t-value is less than 2.5. The graphical representation for
assessment by means of pareto charts in Figure 4.8.a-c. The standardized effects
were studied with pareto charts, which displays as bar graph, the horizontal
values and vertical line specify the
statistically significance at 95% confidence level.
112
Positive and negative signs show the direct and inverse relationship of each
parameter with removal of ofloxacin e.g. pH and time have positive significant
effect on removal of ofloxacin (Fig. 4.8 a-c). Concentration has negative impact on
removal in the case of NaOH and water treated sorbents, however nonsignificant
positive impact was observed in case of HCl treated sorbent. Moreover, amount of
sorbent have significantly positive impact on the removal for all three treated
sorbents. Analysis of variance on these pareto charts (Fig.4.8 a-c) showed p values
lower than 0.05 for all parameters and sorbents other than concentration (p, 0.1007)
in case of HCl treated sorbent. The values of p lower than 0.05 shows the
significance of these parameters for removal of ofloxacin [104]
Figures 4.8 (a-c) and 4.9 (a-c) are generated by Statgraphics software applying
Dapper-Lin composite design as mentioned earlier in the text. The increase in pH
of sorbate enhances the removal efficiency as displayed in Figure 4.8 (a and c) in
case of HCl and NaOH treated sorbents. Adsorption is either a diffusion control,
charge driven or combination of both phenomena. Sawdust is a porous material and
is mainly composed of crude fiber followed by acid detergent, fiber that contains
cellulose and lignin, protein and ash.
113
Figure 4.8 (a-c) Main effect charts for removal of ofloxacin onto treated sawdust (a) HCl,
(b) NaOH and (c) water
The presence of amine, carboxylic functionalities etc. behave as active ion
exchangers. The present carboxylic groups became protonated at lower pH value
(pH ), therefore they became inaccessible to attract positively charged ions
can easily interact with positively charged ions. Ofloxacin is positively charge at
pH lower than 6 and negatively charged above pH 7. The pH behavior (Figure 4.9
a) shows that the removal of ofloxacin by saw dust is not predominantly charge
driven phenomena, therefore it may be suggested that the phenomena is governed
by diffusion of ofloxacin molecule into the pores of sawdust.
114
The optimum sorption conditions determined from mathematical model were
validated by conducting sorption experiment at optimum conditions shows a good
agreement between the calculated and the predicted values for the removal of
ofloxacin in all cases (Table 4.9). However the efficiency of acid treated sorbent
was relatively better than the base and water treated sorbent, therefore, acid treated
sorbent was further characterized to calculate the sorption efficiency.
Table 4.9 (b) Optimum predicted parameter values and model validation
Optimum value (*p) % Removal (*P) % Removal (**E)
Factors HCl NaOH Water HCl NaOH Water HCl NaOH Water
pH 7.5 8.9 3.8 100 77 91.4 95.8 76 91
Amount 50 10 50
Conc. 10 40 48
Time 150 180 70
* P= predictable value, **E= experimental value
4.4.3 Adsorption equilibrium studies
A key factor for adsorbent material is the saturation point for adsorbate onto
adsorbent, known as adsorption capacity. Adsorption isotherms, with their
constants help to determine the adsorption capacity. Hence the Langumir,
Freundlich and D-R isotherms were used to study the equilibrium status for
adsorbate by initial and final response observations. The data was ploted according
to equations below (Eq. 1.1-3).
115
Eq.1.1
Eq.1.2
Eq.1.3
Where qe is the amount of ofloxacin adsorbed onto the surface, Ce is the equilibrium
concentration of ofloxacin in solution. Langmuir, Freundlich and D-R isotherms
were plotted, adsorption capacity denoted by qmax, Kf and KD-R respectively. The
RL calculated as shown in Eq.4.1 RL constant represent the type of acquired
isotherm, RL > 1 presents unfavorable trend, RL = 1 for linear trend observation and
RL = 0 present the irreversible adsorption trend.
Further, KL gives information about binding energy of solute, In Freundlich
isotherm adsorption intensity constant denoted by 1/n. D-
represented polanyil potential, which calculated by term RTln(1+1/Ce) where T is
temperature and R is universal of
adsorption per mole. Table 4.10 (a) shows the obtained constant values by straight
line equations of isotherms.
Eq.4.1
RL values listed in Table 4.10 (a) are less than 1 and greater than zero showing the
favorable nature of adsorption. The values of energy of adsorption calculated from
D-R isotherm found 12.9 K J mol-1.
Table 4.10 (a) Langmuir, Freundlich and D-R sorption isotherm parameters for removal
of ofloxacin onto HCl treated sawdust
Langmuir Freundlich D-R
Q
(mmolg -1)
b×105
(molL-1 )
RL R2 A 1/n
(mmolg-1)
R2 Xm
(mmol g-1)
E
(kJmol-1)
R2
116
0.047 1.04 0.06-
0.58
0.969 3.9 0.471 0.99 0.23 12.9 0.96
4.4.4 Kinetics of adsorption
Mechanism of adsorption of ofloxacin on treated SD adsorbent was investigated by
kinetic study, which was carried out at different time intervals from 1-195 min.
keeping other parameters at their optimum values are shown in Table 4.10 (b). The
experimental data used to evaluate three kinetic models as following: pseudo first-
order, pseudo second order and Morris -Weber by using the equations 3.2 and 3.3,
respectively.
Eq.3.2
Eq.3.3
In the above equations, qe and qt are the adsorption capacities at equilibrium and at
time t respectively. Whereas K1 (min-1) is the rate constant of pseudo-first order of
adsorption, k2 is the equilibrium rate constant for second order rate equation for
kinetics. Intra-particle diffusion (Morris-Weber) model was used to study the mass
transfer resistance on the binding of ofloxacin to the sorbent using Eq.3.4.
Eq.3.4
Where qt is sorption capacity of ofloxacin on sorbent at time t (mg g-1), Kd
intraparticle diffusion rate constant (mg g-1min0.5) and C intercept which gives the
idea of thickness of the boundary layer. Experimental data followed the equation
with correlation coefficient of 0.95. Linear portion of the plot did not pass through
the origin indicating that intra-particle diffusion is not the only rate limiting step. It
supports the idea of complex sorption mechanism with simultaneous intra-particle
diffusion as well as surface adsorption phenomena. Table 4.10 (b) compiles the
data of all three models for adsorption of ofloxacin onto HCl treated sawdust. The
117
experimental qe values and theoretical qe values were approximately same for all
selected kinetic models, also, the calculated correlation coefficients, R2 obtained
from pseudo-second-order kinetic model were closer to unity as compare to the
pseudo-first order kinetic model. These observed outcomes show the pseudosecond
order adsorption process is predominant in current adsorption case.
Table 4.10 (b) Kinetic parameters for removal of ofloxacin onto HCl treated sawdust
Pseudo first order Pseudo second order Moris-Weber
K1, min-1 qe
mgg- 1
R2 K2 µgg-1 min-
1
qe mgg- 1
R2 Kd µgg-1min-1 R2
0.0152 5.55 0.924 0.0073 10.07 0.997 0.24 0.950
4.4.5 Thermodynamics
Thermodynamic study helps in estimating the feasibility of the sorption process.
The temperature effect on the sorption of ofloxacin was carried out at range of
temperatures, 25-50ºC followed by optimum condition of sorption. By plotting
lnKC versus 1/T (T in Kelvin) obtained the linear plot for sorption experiment.
y the
following equations (Eq4.2-4).
Eq.4.2
, Eq.4.3
G RT lnKC Eq.4.4
Fe represents the fraction of ofloxacin sorbed at equilibrium. From the plot of lnK
versus 1/T, the correlation factor was used as 0.95 to estimate the values of H =
118
40.5± 1.63 kJ mol 1, S = 119 ± 5.62 Jmol 1 K 1, and G (303K) 6.28 ± 0.11 KJmol1.
The obtained values of H and G were negative specify the exothermic and
spontaneous nature of sorption.
4.4.6 Application of removal process
The real water samples were analyzed using reported procedure by spiking 100 mL
of sample with 100 ng mL-1 of OFL. All samples (neat and spiked) were cleaned
prior to chromatographic determination using solid phase extraction. The solid
phase extraction method was adopted from method reported by Gros et al [98]. In
order to establish the reliability of the reported method extraction of ofloxacin,
recovery experiments were carried out. Apparent recoveries, calculated as the ratio
of the measured concentration of standards levels to the spiked synthetic
wastewater (expressed as percentage). For recovery, known amounts of ofloxacin
added whose concentration after pre-concentration reached at 1, 5 and 10 µg mL-1;
the observed recoveries were 77%, 84% and 94%, respectively.
Figure 4.9 HPLC chromatogram for spiked hospital wastewater sample ofloxacin
(10 µg mL-1) (a) and after removal (b)
119
Figure 4.9 shows a chromatograph of real water sample and water sample spiked
with standard ofloxacin. The obtained results are shown in Table 4.11 which shows
that removal efficiency was in the range of 84.9 to 89 %. The presence of
IBP found in ng mL-1 level in nearly all of the samples which is higher as
compared to earlier reports, mainly due to inefficient waste treatment plants in
locality.
Table 4.11 Removal of OFLX in hospital wastewater water samples followed by
SPE
Spiked sample* % Removal
sample A 85.4
sample B 89.0
sample C 87.3
sample D 84.6
*Each sample was spiked with 100 ng mL-1 prior to preconcentration step.
120
4.5 Removal of ciprofloxacin onto treated peanut shells (Arachis
hypogaea L.) by response surface methodology (RSM)
Ciprofloxain (CIP) is second generation antibiotic, contains a piperazine
group at position7 of the 4-quinolone nucleus in structure. The zwitterion behaved
CIP from fluoroquinolone antibiotic is considered as environmental contaminant
[127, 130].
The removal of ciprofloxacin was accomplished by agricultural waste bio product
peanut shells (PS) after treated with acid (HCl) and alkali (NaOH). The main
compositions of PS are cellulose, hemicellulose and lignin [212]. Peanut shells
were treated with alkali (NaOH) and acid (HCl) for sorbent preparation.
Ciprofloxacin (CIP) from aqueous solution by RSM using CCD to optimized the
removal efficiency. Set of 18 experiments was used and factors such as pH, amount
of sorbent, contact time and concentration of sorbate were considered the critical
factors to study the CIP removal.
4.5.1 Mathematical and statistical procedure for CIP removal
To aim the efficient removal of ciprofloxacin onto treated PS, Face Centered Diper-
Lin small composite design of 18 experiments was used. The range and level of
experimental variables are shown in Table 4.12 (a).
Table 4.12 (a) Variables code levels of design used in experimental for removal of
CIP on treated PS
Independent variable Code levels
-1 0 +1
pH, A 2 6 10
Concentration (mg L-1), B 5 52.5 100
Time (min.), C 10 65 120
Amount (mg), D 50 150 250
121
All experiments were performed in triplicate and random order. The response
function coefficients were determined by regression using computer software
Statgraphics Centurion (XVI) from Statpoint Technologies, Inc, USA.
4.5.2 Experimental design
Daper-Lin composite design was used in this study to determine the correlation
between aqueous solution variables and sorption efficiency of CIP on acid and
alkali treated PS. The results obtained from the design are shown in Table 4.12
(b).
The maximum percent sorption observed for acid treated PS and alkali treated PS
were 98.7% and 95% respectively. Capacity for acid treated PS was 21.5mg/g and
alkali treated PS capacity was found to be 15.7mg/g, acid treated sorbent shows
higher capacity for CIP sorption than alkali treated sorbent. CIP is zwitterionic in
nature, particularly in wastewater due to its ionic nature, evidently reported with
two pka values; inonization of carboxylic at pka value 6.16 and piperazinyl N-4 atom
at pKa 8.63.9 [213].
4.5.3 Statistical analysis for CIP removal
Regression analyses were performed for both acid treated PS and alkali treated PS
for CIP sorption. Regression analysis was aimed to fit the response function of
CIP sorption on treated PS. According to obtained P-value in case of acid treated
PS as compare to alkali treated PS variables are highly significant, in case of
Residual plots for observed response and fitted response for the acid and alkali
treated PS sorbents were plotted as shown Fig. 4.10 (a) and (b) respectively.
122
Table 4.12 (b) Experimental %sorption and predicted %sorption of ciprofloxacin
HCl Treated NaOH Treated
set of
experiment
Observed
value
Fitted
value
± SD Observed
value
Fitted
value
± SD
1 59.0 59.1 ± 1.03 35.9 35.3 ± 2.6
2 50.2 46.2 ± 3.1 46.9 48.4 ± 1.2
3 74.9 75.9 ± 0.2 37.6 37.6 ± 5.2
4 63.6 64.6 ± 1.2 51.6 55.7 ± 0.64
5 96.0 97.0 ± 2.6 95.0 98.0 ± 0.82
6 48.0 46.2 ± 3.3 44.1 48.4 ± 0.2
7 76.8 77.8 ± 2.5 37.9 42.0 ± 0.61
8 83.9 83.9 ± 1.2 32.5 35.9 ± 0.2
9 48.9 50.9 ± 2.5 41.6 41.3 ± 1.2
10 91.7 91.2 ± 0.18 91.5 91.1 ± 1.42
11 23.7 22.7 ± 3.2 4.7 4.7 ± 0.18
12 98.7 98.7 ± 0.43 92.0 93.0 ± 3.3
13 83.5 83.5 ± 5.2 47.7 47.1 ± 0.29
14 34.1 35.1 ± 3.5 5.6 6.6 ± 3.2
15 71.8 71.8 ± 2.2 46.6 46.0 ± 0.25
16 78.3 77.8 ± 5.2 44.1 44.6 ± 5.3
17 87.8 87.3 ± 1.2 43.9 43.4 ± 0.21
18 49.3 48.8 ± 1.03 4.2 4.3 ± 0.82
To evaluate the calculated effects were significantly different from zero, Students
t test was done at 95% of confidence level and eight degrees of freedom. Sorption
of CIP onto acid treated PS obtained t value was 0.768, and 0.754 for alkali treated
PS sorbent Figure 4.10 (c&d). Pareto chart for acid treated PS and alkali treated PS
123
for CIP, pH is highly significant variable effect for both sorbents and also with
positive correlation to %sorption.
Figure 4.10 (a &b) Residual plots of ciprofloxacin sorption onto acid treated PS
(a) and alkali treated PS (b)
124
Figure 4.10 (c&d) Pareto charts for acid treated PS (c) and alkali treated PS (d)
Concentration has negative correlation to %sorption, highly significant factor for
acid treated PS as compared to alkali treated PS. Concentration and amounts
interactive effect is significant for both, acid treated PS and base treated PS with
positive correlation to % sorption. Three dimensional response surface plots for
uptake of CIP by acid treated PS and alkali treated PS are given in Fig. 4.10 (e) and
(f) respectively.
d
125
pH µg mL-1 pH µg mL-1
Figure 4.10 (e & f ) Response surface plots for CIP sorption onto acid treated PS
(e) and alkali treated PS (f)
The obtained optimum values for acid treated PS and alkali treated PS are shown
in table 4.12 (C), for further studies variable factors were kept at optimum levels
presented by software calculations.
Table 4.12 (c) Optimum predicted parameter values and model validation
126
Factors Optimum Value % Removal (*P) % Removal (**E)
HCl NaOH HCl NaOH HCl NaOH
Amount(mg) 250 100 97 100 96 95.02
pH 8 9.7
Con. 10 35
(µg mL-1)
Time(min.) 77 71
*P=predicted by software; **E= Experimental obtained response
4.5.4 Adsorption kinetics and isotherm
The equilibrium sorption of CIP was carried out by enclosing treated PS sorbent, contained
20 mL of volume with different concentrations of CIP (5 mg L-1 100 mg L-1) in thermostat
rotary shaker at 30 ºC with speed of 120 rpm. The mixture was filtered and the filtrate was
run on HPLC-UV for remaining CIP concentrations. The obtained sorption data was
analyzed for each one of isotherms linear equation are shown in Table 4.14(a). Langmuir
isotherm model was dominant for acid treated PS, however for alkali treated PS Freundlich
isotherm model is well fitted but the obtained mean sorption energy (E) by D-R isotherm,
is under the magnitude of the physisorption process. Possibly, the treated PS showed
heterogenous sorbent surface behavior trend due to different interaction strengths and
adsorption energy of CIP and treated PS. The maximum sorption capacities were observed
by Langmuir isotherm were 42.2 µmol g-1 and 10.12 mmol g-1 onto acid treated PS and
alkali treated PS, respectively . The acid treated PS shows better adsorption capacity than
the alkali treated PS. CIP adsorption shows a maximum at pH 8 because of the electrostatic
interaction between positively charge CIP and negatively charged acid treated PS.
The kinetic data fit well with the pseudo-second order and the pseudo-first order
model for CIP sorption onto acid treated PS. For alkali treated PS sorbent
pseudosecond order of kinetics is dominant as compared to rest two kinetic models.
For both acid treated PS and alkali treated PS the sorption of CIP onto PS followed
pseudo-second order model with correlation of coefficient (r 1).
Table 4.13 (a) Fitting parameters for Langmuir, Freundlich and D-R isotherm
models for adsorption of ciprofloxacin onto treated PS
127
Sorbent
Treatment Langmuir Isotherm
Q b
(µmol/gram) (L/mol) RL R
Alkali
treated 10.12 12
0.85-
0.96 0.9
Acid
treated 42.2 9.2
0.56-
0.9 0.99
Freundlich Isotherm
Kf (mmol/g) 1/n r
Alkali
treated 1.73 1.05 0.97
Acid
treated 2.5 0.614 0.91
D-R Isother m
Xm (mmol/g) E r
Alkali
treated 1.109 9.12 0.95
Acid
treated 2.3 7.7 0.9
Table 4.14.(b). Pseudo-first order kinetic model, pseudo-second order model, and
morris-weber kinetic model parameters for adsorption of sorption of CIP onto
treated PS
Acid treated PS Alkali treated PS
Pseudo first order
K1, min-1 0.0043 0.00092
qe mmolg-1 0.146 0.111
r 0.997 0.82
Pseudo second order
K2 molg-1 min-1 5.07x 10-5 1.9
qe mmolg
-1 5 2.2
r 0.999 0.999
Moris-Weber
128
Kd mmolg-1min-1 0.1 0.2
r 0.98 0.81
4.5.5 Surface characterization
Scanning electron micrographs (SEM) of acid treated PS samples are shown in Fig.
4.11 a and b. It is clear from the SEM image; the external surface is rough and
concentrated with microporous structures on surface of different shapes and sizes
were observed. The BJH model obtained surface area (818.52 m2 g-1) is due to the
presence of micropores, while the contribution of mesoporosity (40 m2 g-1) is
relatively small (Figure 4.12).
Figure 4.11 (a& b) SEM images of acid treated peanut shells at 20 µm (a) and
50 µm (b)
129
Figure 4.12 N2-adsorption and desorption isotherms at 77K corresponding BJH pore-size
distribution
4.5.6 Application of removal of CIP from Hospital wastewater onto acid treated PS
Prior to CIP batch sorption at optimized conditions all five hospital wastewater
samples 20 mL of each sample was spiked and filtered with 0.45µm filter paper.
The filtrated samples were run for batch process for sorption of CIP onto acid
treated PS, results are shown in Table 4.15.
Table 4.15 Spiked hospital wastewater samples removal by acid treated PS
Spiked Conc.(µg mL-1) % Removal ± S. D
5 86.8 ± 0.37
20 78 ± 0.42
100 76.8 ± 0.34
130
Figure 4.13 Hospital wastewater spiked sample 20 µg/mL (a) and after batch sorption response (b)
131
4.6 Simultaneous removal of Ibuprofen and Diclofenac sodium onto
HTC derived from ber fruit (Ziziphus mauritiana L.) by RSM
Hydrothermal carbonization of Z. Mauritiana L. (HTC-ZM) was prepared
from crushed whole ber fruit and heated in closed autoclaves in the presence of an
acidic catalyst (citric acid) to 200 C for 16 hour. Acid catalyst helps in the
suppression of gas formation and leads to more controlled decomposition. Using
these conditions, gas formation is shifted to dehydration reactions and coalification
instead of hydrid transfer and decarboxylation [207]. (HTC-ZM) under acidic
conditions was yielded 60% of brown colored carboneous material, which is much
greater in yield than the obtained for orange peels under similar conditions [214].
Authors reported the yield for orange peels as 37% this may be due to
compositional difference of the two materials in terms of sugars, cellulose,
hemicelluloses and lignin content and carbonization conditions [215]. Final yield
of hydrothermal carbonized Z. Mauritiana L. (HTC-ZM) was based on filtered off
product calculated by Eq. 3.5., which was washed several times (distilled water and
absolute ethanol), then finally dried in an oven at 80 °C for 4 h.
4.6.1 Surface characterization
Surface functionalities of HTC- ZM were determined from FTIR. The obtained
spectra showed band from 3500 to 3200 cm-1, which is attributed to OH stretching
vibrations. Peak at 2927 cm-1 shows CH alkane stretching vibrations while peaks at
1710 cm-1 and 1600 are characterized for C=O and C=C (carbonyl, quinone, ester
or carboxylic functionalities) respectively. At 1030 cm-1 peak represents CO-C
stretching vibrations and peak at 804 cm-1 represents C-H aromatic out of plane
bending vibrations. HTC-ZM showed typical signature functionalities of
hydrochar. The pattern of bands in IR spectra are similar to HTC obtained from
glucose and starch [207, 216]. IR spectra indicate that HTC-ZM surface is polar
(Figure 4.14 a).
132
Figure 4.14 (a) FTIR-ATR of prepared HTC-ZM
SEM micrographs of HTC -ZM at 10 µm scale are presented in Fig 3 (a) which
SEM images ( Figure 4.14 b & c) shows aggregated particles in clumps of various
sizes. Such aggregation of particle may arise from the typical carbonization
mechanism of lignocellulosic biomass which involves different path than the pure
glucose or water soluble substrates. Under these conditions, the cellulosic substrate
undergoes intramolecular condensation, dehydration and decarbonylation reactions
leading to the production of a hydrothermal carbon structurally composed of a more
condensed polyaromatic arene-like carbon domains instead of formation of
hydroxyfurfuralaldehyde when glucose is used as substrate [215]. Such, aggregated
structures are typical for HTCs obtained from lignocellolosic biomasses. Fig. 4.15
(c) shows zoomed-in portion at 5 µm scale which shows that average particle size
is around 1µm. Particle size bigger than 500 nm is normally seen at higher
temperature (above 180 C) and for longer reaction time. Herein, reaction was
carried out at 200 C and for 20 hours therefore, such particle sizes are expected.
Surface of particles is not smooth which indicates that particles are porous in nature.
Typical elemental composition of HTC-ZM was found to be carbon (67.3%),
hydrogen (4.34%), nitrogen (3.19) and oxygen as found by difference was
24.05%.
133
Figure 4.14 (b & c) SEM images of HTC-ZM at 10µm (b) and 5 µm (c)
Surface area obtained through nitrogen adsorption porosimetry using BJH model
was found to be 1160 m2 g-1, which is better than obtained under similar acidic
conditions for orange peels [214]. This may be due to washing of hydrochar with
ethanol which removes trapped colloidal carbon in the pores of hydrothermally
carbonized material [217].
Figure 4.15 N2-adsorption and desorption isotherms at 77K corresponding BJH
pore-size distribution
4.6.2 Adsorption studies
134
Characterization of HTC-ZM reveals that the hydrochar obtained from fruit of Z.
Mauritiana L. under acidic conditions and washed with ethanol resulted in porous
carbonized material with polar oxygen functional groups on surface. Therefore, it
is suitable to be tested as sorbent for removal of contaminants from aqueous
systems.
To optimize removal of the two drugs, IBP and DFS, onto HTC-ZM, statistical
experimental design was used instead of classical one parameter optimization
methodology, which reduces the number of experiments while providing good
insight into optimum parameters. The code levels of CCD are shown in Table
4.16 (a). The results obtained from the design are shown in Table 4.16 (b).
Table 4.16 (a) Levels of factors used in experimental design for removal of DFS
& IBP onto HTC-ZM
Amount (mg) 50 150 250
Concentration (µgL-1) 10 55 100
pH 2 5 8
Time (min) 10 65 120
4.6.3 Optimization of IBP & DFS simultaneous removal by factorial design
The R-Squared statistic indicates that the model as fitted explains 98.73% of the
variability in % sorption. The observed P-value is less than 95.0%, which is an
indication of possible serial correlation at the 95.0% significance level.
The residual plots of HTC sorbent for IBP and DFS (Fig. 4.16 a-b) show a fairly
random pattern with scattered values around the axis. Random dispersion of values
around the horizontal axis validates the fitness of linear regression model. Effects
of various reaction conditions like pH, amount of HTC-ZM, concentration of drugs
and contact time that may affect adsorption of DFS and IBP are explained through
0 1
135
'main effect plot' generated by Statgraphics software applying Dapper-Lin
composite design as mentioned earlier in the text.
Table 4.16 (b) Experimental %sorption and predicted %sorption of diclofenac
sodium (DFS) and ibuprofen (IBP) onto HTC-ZM
IBP DFS
Set of
experiment
Observed
%sorption ± SD
Fitted
%sorption
Observed
%sorption ± SD
Fitted
%sorption
1 62.5 ± 1.2 62.2 69.3 ± 0.18 70.0
2 90.4 ± 4.1 90.5 59.5 ± 3.2 60.1
3 39.4 ± 2.21 39.1 51.3 ± 0.43 52.0
4 48.2 ± 5.3 48.0 44.3 ± 5.2 44.9
5 97.1 ± 4.1 97.2 89.3 ± 3.5 90.0
6 94.0 ± 5.5 94.5 66.7 ± 2.2 67.0
7 61.0 ± 1.7 61.5 65.1 ± 5.2 65.7
8 75.6 ± 2.2 75.7 72.5 ± 1.2 73.2
9 51.8 ± 1.2 52.3 54.8 ± 1.42 54.7
10 77.7 ± 1.42 78.2 78.7 ± 2.6 78.6
11 73.6 ± 2.6 73.4 78.7 ± 1.2 79.4
12 44.1 ± 1.2 44.9 60.9 ± 2.5 60.9
13 64.0 ± 5.2 64.8 70.5 ± 0.18 70.5
14 61.3 ± 0.64 61.5 65.8 ± 3.2 65.7
15 50.0 ± 0.82 51.0 64.3 ± 0.43 64.3
16 3.2 ± 0.2 3.3 86.0 ± 5.2 87.0
17 51.3 ± 0.61 51.2 60.7 ± 3.5 61.4
18 3.3 ± 1.5 3.5 37.0 ± 0.45 37.0
136
The main effect plots for both IBP and DFS (Figure 4.16 c & d) show that
increasing amount of HTC-ZM increases the adsorption which reaches at 86% and
92% for DFS and IBP respectively with 250 mg of sorbent. Also, increasing contact
time more than one hour does not affect the sorption, however, pH and
concentration of drug have different effect for the two drugs.
Figure 4.16 (a & b) Residual plots for (a) diclofenac sodium and (b) Ibuprofen
sorption onto HTC-ZM
137
For DFS at lower and higher pH values adsoprtion is not favourable where so for
IBP acidic pH favors adsorption whereas at higher pH sorption decreases. This may
be due to negative charges on surface of sorbent and both the drugs dominates the
electrostatic repulsion and reduces favorable interactions whereas in the case of
DFS lower adsorption at pH < 4 may be due to intermolecular cyclization [218]
which changes the nature of the molecule. Concentration profile indicates that
HTC-ZM has better accessible adsorption sites towards IBP than DFS; it is
reflected from concave type curve for DFS while increasing adsorption at lower
concentration for IBP showed maximum %sorption.
Figure 4.16 (c & d) Main effect plot of % sorption of (c) DFS and (d) IBP onto
HTC-ZM
The 3D response surface plots (Fig.4.16 e & f) are also plotted. These 3D response
graphs show the combined effect of solution pH and sorbent amount on %sorption
of drugs keeping other parameters at their optimized conditions.
138
pH
(mg)
Figure 4.16 (e &f) Response surface plot for sorption of IBP (e) and DFS (f) onto
HTC-ZM
The optimum % sorption conditions obtained from CCD were validated by
experiments carried out at optimum conditions (Table 4.17). A maximum
experimental sorption for IBP and DFS were obtained 97% and 90%, respectively,
which is close as compare to values predicted from CCD. This also proves
reliability of experimental design. Further, all other experiments were performed at
amount of sorbate (250 mg), pH (4.0), concentration of sorbent (10 mg L-1) and
contact time (88 minutes).
139
Table 4.17 Design validation for DFS & IBP removal by comparing design
predicted and obtained experimental response onto HTC-ZM
Factors Optimum Value
(*P)
% Removal (*P) % Removal (**E)
IBP DFS IBP DFS IBP DFS
Amount 250 250 100 90 97 88
pH 4 4.4
Con. 11 10
Time 88 80
*P = predicted value **E= experimental value
4.6.4 Sorption isotherms
The sorption isotherm is a preliminary step to evaluate diversity of sorbent and
sorbate in sorption process. The adsorption isotherms were determined with
different IBP and DFS solution concentrations ranging from 5 to 200 mg L-1 for 80
min. The initial pH of all the solutions used in the sorption was kept at pH = 4. The
samples were withdrawn from a thermostated shaker and immediately filtered to
determine adsorbed IBP and DFS concentrations by HPLC-diode array detector
(DAD). All the experimental treatments were performed in triplicate and the
average values are reported. The observed results are shown in Table 4.6.4.
Evaluation of the three isotherms obtained findings are given in Table 4.18 (a). It
was observed that Dubinin-Radushkevich (D-R) isotherms and Freundlich
isotherms were showed correlation coefficient closer to unity with well fitted data.
Pore diameter of HTC-ZM as obtained through nitrogen adsorption porosimetry
was 75 ºA, which shows that it is mesoporous in nature. Mesoporous sorbents
shows complex adsorption behavior. Consequently, the monolayer and multilayer
adsorption phenomenon presumed onto HTC-ZM where molecules can fill the pore
by sticking on the walls (active sites), form multilayeres followed capillary
condensation. Adsorption in mesoporous material is supported by D-R isotherm on
the basis of pores filling theory [219]. From linear plot of D-R isotherm, adsorption
capacity of HTC-ZM was found to be 2.03 mmol gram-1 for DFS and 2.54 mmol
140
gram-1 for IBP. The mean free energy for both, DFS and IBP 8.1 KJ mol-1 and 8.3
KJ mol-1respectively, which indicates a physiosorption process.
Table 4.18 (a) Fitting parameters of Langmuir isotherm, Freundlich isotherm and
D-R isotherm for sorption equilibrium of diclofenac sodium and ibuprofenn
Drug Langmuir Isotherm
b
Q RL
(L/mol)
(mmol/gram)
r
Diclofenac
sodium 0.28 6.2 0.088-0.54
0.9
Ibuprofen
0.12
Freundlich
7.6 0.029-0.42
Isotherm
0.93
Kf (µg/gram) 1/n
r
Diclofenac
sodium 513 0.98 0.95
Ibuprofen 281 1.021 0.96
D-R Isotherm
Xm
(mmol/gram) E r
Diclofenac
sodium 2.03 8.1 0.97
Ibuprofen 2.54 8.3 0.99
4.6.5 Kinetics study
The Kinetics of DFS and IBP sorption were determined from the time versus
%removal plots. The rate kinetics of DFS and IBP on HTC-ZM was analyzed by
using 1st-order rate equation, 2nd order rate equation and Morris-Weber rate
equation (section 4.4.4)
141
Table 4.18 (b) shows that the values of regression coefficients in all cases were
above 0.9 which reflects the goodness of fit however at higher concentrations; 3.14
10-4 M and 4.85 10-4 M for DFS and IBP, respectively errors in the
regression coefficient were observed. Also, the values of rate constant K2 for
second order kinetics using higher concentrations were practically (rate is 2.9 and
5.8 mol g -1 min-1) not possible. Adsorption of both drugs followed equations, first
order and second order kinetics which shows that sorption is governed through
multiple interactions. Diffusion coefficient obtained through Morris-Weber
equation for IBP were higher than DFS while the plot of vs qt does not pass
through origin reflects that diffusion is not purely film diffusion but mixed.
Table 4.18 (b) Kinetic parameters for removal of DFS and IBP onto HTC at
concentration level of 3.14 10-5 M, (4.85 10-5 M), respectively. Constant shown
in parenthesis indicates higher concentrations of drugs; 3.14 10-4 M and
-4
R2 0.99 (0.96) 0.99 (0.98)
Pseudo second order
R2
Morris-Weber
0.999 (0.996) 0.998 (0.998)
Kd, mol g-1min-1 2 10-5 (6 10-5) 4 10-5 (2 10-3)
10
1 1 4.61
10
6.91 10
10
q 1 8.03 10
10
10
10
2 , 1
1 6.9 10
10
q 1 2.1 10
10
10
10
142
Passes
origin
through Deviates much from
origin
Deviates much from
origin
R2
0.95 (0.97) 0.98 (0.98)
4.6.6 Removal from synthetic wastewater
Adsorption of DFS and IBP with RSM obtained optimum conditions was employed
to synthetic wastewater, spiked with IBP and DFS both drugs at three different
initial concentrations (5 mgL-1, 10 mgL-1and 50 mgL-1). The obtained percent
removals were observed from 67% to 86% as shown in Table 4.19.
Table 4.19 Synthetic wastewater removal of spiked IBP and DFS onto HTC-ZM
Drug
Spiked
Conc.(µg/mL)
%
Removal ±SD
IBP 5 86.8 ±0.37
DFS 5 80.2 ±0.55
IBP 10 78 ±1.2
DFS 10 76.5 ±1.37
IBP 50 73.3 ±1.24
DFS 50 67 ±1.39
143
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion
This study concludes that hospital wastewaters in Hyderabad, Pakistan are
contaminated with PhACs. This is matter of concern as these hospital waters enter
into main canals (water distributaries used for drinking and irrigation purposes)
without treatment.
LC-MS, GC-MS and HPLC techniques were found adequate for detection of
PhACs residues. Clean-up and preconcentration using solid phase extraction leads
to lower detection limits for residual drugs in environmental waters. Two simple
methods were developed during current study using SPE were successfully applied
to determine PhACs in hospital wastewaters.
Residues of quinolone antibiotics, anti-inflammatory and analgesics were detected
- lactam antibiotics were
not
-lactam group of drugs were studied
further and observed that these degrade faster in alkaline conditions and even at
room temperature. Absence of this group of compounds in hospital wastewater may
be due to fast degradation of this group in aqueous conditions. Further studies may
be carried out on the environmental degradation products of this as well as other
groups of drugs. It is advisable to remove these drugs from hospital wastewaters at
the discharge points. This will reduce the residence time for the drugs to produce
unknown degradation products which otherwise would be even difficult to treat.
144
Removal of drugs can be simply carried using adsorption technique employing bio-
sorbents. Current study shows that thermal or mild basic treatments can produce
efficient sorbents for removal of these drugs. Among various sorbents studied most
of them showed excellent removal efficiencies. Kinetics and isotherm models
showed physisorption phenomenon for uptake of drugs on biosorbents.
To effectively utilize the results of this study, a continuous flow operated column
adsorption studies and scalability of biosorbent based hospital wastewater process
is suggested here for further study.
5.2 Recommendations
The hospitals effluent drains are continually entering into water streams without
any efficient remediation into River Indus, Pakistan; this is the rooting cause of
water quality depletion. The Indus River is the major water source for large number
of population. It is important to preserve the quality of Indus River. The presence
of these pharmaceuticals active compounds can be eliminated by the following easy
three steps.
o Prevention:
Proper disposal of unused drugs
Public awareness campaigns to educate the public o
Regulation:
Proper check and balance of pharmaceutical compounds circulation all
over the places, where pharmaceuticals are consumed or sold.
Pharmacies and hospitals may be advised and provided information on
proper disposal of chemicals and drugs.
o Remediation: Hospital effluent need to be treated with advanced technologies
for removal of active pharmaceuticals compounds.
145
