applicability of biomaterials for the recovery of platinum...

Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of

International Master of Science

in Environmental Technology and Engineering

an Erasmus Mundus Master Course

jointly organized by UGent (Belgium), ICTP (Prague) and UNESCO‐IHE (the Netherlands)

Academic year 2013 – 2014

Applicability of biomaterials for the recovery of platinum

species from urine

Ghent University, Belgium

Alebel Abebe

Promoter:

Prof. dr. ir. Gijs Du Laing

This thesis was elaborated at Ghent University and defended at Ghent University, Belgium within the

framework of the European Erasmus Mundus Programme “Erasmus Mundus International Master of Science in

Environmental Technology and Engineering” (Course N° 2011-0172)

© 2013-2014, Ghent, Belgium, Alebel Abebe, Ghent University, all rights reserved.

I

Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of

International Master of Science

in Environmental Technology and Engineering

Applicability of biomaterials for the recovery of platinum

species from urine

Ghent University, Belgium

Alebel Abebe

Promoter:

Prof. dr. ir. Gijs Du Laing

Tutor:

Karel Folens

This thesis was elaborated at Ghent University and defended at Ghent University, Belgium within the framework of the European Erasmus Mundus Programme “Erasmus Mundus International Master of

Science in Environmental Technology and Engineering”

II

Copy right

This M.Sc. thesis work is not published and not prepared for further distribution. The author

and the promoter give the permission to be used for consultation purpose and to copy parts of

it for personal use. Every use other than this is subject to the copyright laws; more

specifically the source must be extensively specified when using results from this thesis.

© Ghent University, August 2014

The Promoter The Author

Prof. dr. ir. Gijs Du Laing Alebel Abebe

III

Acknowledgements First of all, I would like to convey my appreciation and gratefulness to my advisor Prof. dr. ir.

Gijs Du Laing for the unreserved support and guidance he did during the entire thesis work

from the title selection until the end. I really appreciate his motivation, enthusiasm and

immense knowledge on the subject that helped me a lot to bring the thesis to the standard. His

tireless work during finalization of the thesis was superb.

I also would like to extend my deep gratitude to Karel Folens for his close supervision,

guidance, discussion, and analysis of every experimental work. He was so supportive,

welcoming, friendly, and nice to me during the whole thesis work in general and the

experimental set up and analysis in particular. I could not have imagined having a better tutor

for my thesis.

The European Commission of Education, Audiovisual and Culture Executive Agency is

highly credited for giving me the opportunity to get the knowledge and experience in the

European leading universities and enjoy wonderful academia.

My special thanks also addressed to the people working in the eco-chemistry lab. I thank you

Roseline, Joachim and Katty. The best lab atmosphere I have ever experienced and I really

learned a lot how to help others when they are in need. A kind of people always ready for

help and it was a great pleasure working with you.

My sincere appreciation also goes to Dolores Esquivel in the department of inorganic and

physical chemistry of Ghent University for providing the PMO-SH and PMO-SH(50%) sorbent

with detail preparation and characteristics.

I also very rarely I have felt that I am staying away from home due to the presence of my

brothers and sisters around me that make my stay so wonderful and gave a great experience.

And my exceptional thanks goes to my mom who thought me endurance and hard working

that ultimately plays role to my current achievements. Thanks for being a wonderful mother.

IV

Table of Contents Acknowledgements ...................................................................................................... III Table of Contents ........................................................................................................ IV List of Figures ............................................................................................................. VI List of Tables .............................................................................................................. IX

Abstract ...................................................................................................................... XI Acronyms ................................................................................................................. XIII 1. Introduction .......................................................................................................... 1

2. Objectives ............................................................................................................. 3

2.1 General objective ............................................................................................. 3

2.2 Specific objectives ........................................................................................... 3 3. Literature review ................................................................................................... 4

3.1 History of platinum use, its sources and release in to the environment ................... 4 3.2 Cancerostatic platinum compounds (CPC) .......................................................... 7

3.2.1 Platinum containing chemotherapy ............................................................. 7

3.2.2 Mechanism of action of CPC ...................................................................... 9

3.2.3 Exposure route .......................................................................................... 9 3.2.4 Environmental and human health impact of CPC .......................................... 9 3.2.5 Contribution of hospitals towards platinum concentrations in municipal

sewage... ............................................................................................................. 10

3.3 Options for platinum removal and recovery from wastewater ............................. 11

3.3.1 Biosorption ............................................................................................. 11

3.3.2 Biomaterials used for sorption of platinum ................................................. 12 3.4 Factors affecting biosorption ........................................................................... 15

3.5 Sorption models and isotherms ........................................................................ 16 4. Materials and methods ......................................................................................... 18

4.1 Introduction .................................................................................................. 18

4.2 Variables ...................................................................................................... 18

4.3 Materials ...................................................................................................... 18

4.3.1 Synthetic urine ........................................................................................ 19 4.4 Methods ....................................................................................................... 19

4.4.1 Batch adsorption study............................................................................. 19

4.4.2 Fixed bed adsorption study (column test experimental set up) ...................... 22

4.4.3 Desorption experiment ............................................................................. 24 4.4.4 Characterization of biomaterials ............................................................... 24 4.4.5 Pt analysis .............................................................................................. 25

4.4.6 Quality control ........................................................................................ 25 5. Results ............................................................................................................... 26

5.1 Characterization of the biomaterials ................................................................. 26

5.2 Batch adsorption studies ................................................................................. 26 5.2.1 Sorption to biomaterials ........................................................................... 26

5.2.2 Sorption to PMO-SH ............................................................................... 35 5.2.3 Effect of concentration on sorption ........................................................... 36

5.2.4 Fixed bed adsorption study (continuous flow test) ...................................... 41 5.2.5 Column optimization ............................................................................... 44

5.2.6 Full scale column experiment ................................................................... 48 5.2.7 Desorption study ..................................................................................... 51

6. Discussion .......................................................................................................... 52

V

6.1 Introduction .................................................................................................. 52 6.2 Screening of biomaterials ............................................................................... 52

6.3 Potential use of materials for Pt sorption .......................................................... 52 6.3.1 Biomaterials ........................................................................................... 52

6.3.2 PMO-SH ................................................................................................ 56 6.4 Sorption isotherm .......................................................................................... 57

6.5 Sorption test on continuous flow system ........................................................... 58 6.6 Desorption experiment ................................................................................... 60 6.7 Economic evaluation ...................................................................................... 60

7. Conclusion .......................................................................................................... 63 8. Further research ................................................................................................... 65

9. References .......................................................................................................... 66

Annex- I: Post Hoc test result of Tukey HSD to examine differences or similarities in

sorption between biomaterials for each Pt species at pH 7; test was done after

homogeneity of variance was proven with P-value > 0.05 ........................................ 72

Annex-II: Post Hoc test result of Tamhane to examine significant differences or

similarities in sorption of [PtCl6]2-

between tested pH levels for each biomaterial

(activated carbon (AC), chitosan and biochar); test was done after test for variance

homoginity failed with P-value < 0.05 ................................................................... 73

Annex-III: Post Hoc test result of Tamhane to examine differences or similarities in

sorption of cisplatin between tested pH levels for each biomaterial (activated carbon

(AC), chitosan and biochar); test was done after test for variance homogeneity failed

with P-value < 0.05 .............................................................................................. 77

Annex-IV- Post Hoc test result of Tukey HSD to compare differences or similarities in

sorption between different Pt species by using dual bed column packed with activated

carbon and chitosan; test was done after homogeneity of variance was proven with P-

value > 0.05 ........................................................................................................ 79

VI

List of Figures

Figure Page

Figure 1: Concentrations of platinum and palladium in ppb for different environmental

samples in Sheffield, UK....................................................................................................6

Figure 2: Structural formula of three platinum derivatives.......................................................8

Figure 3: Structure of chitosan.................................................................................................12

Figure 4: Columns containing biomaterials for continuous adsorption study..........................23

Figure 5: TOC released by biomaterials using synthetic urine at pH 6, after 24h shaking at 25 oC (mean + standard deviation, n=3).................................................................................26

Figure 6: Relative removal of [PtCl6]2-

, cisplatin, carboplatin and oxaliplatin by activated

carbon, chitosan and biochar at pH 7 using synthetic urine (mean + standard deviation,

n=3). The initial Pt concentration was 100 µg L-1

...........................................................27


by different

combinations of biomaterials (in a 50-50

mixture ratio) using synthetic urine at pH 7 (mean + standard deviation, n=3). The initial

Pt concentration was 100 µg L-1

......................................................................................28


by activated carbon, chitosan and biochar at different

pH levels using synthetic urine (mean + standard deviation, n=3). The initial Pt

concentration was 100 µg L-1

............................................................................................29

Figure 9: Relative removal of Cisplatin by activated carbon, chitosan and biochar at different

pH levels using synthetic urine (mean + standard deviation, n=3). The initial Pt


............................................................................................31

Figure 10: Relative removal of carboplatin by activated carbon, chitosan and biochar at

different pH levels using synthetic urine (mean + standard deviation, n=3). The initial Pt


............................................................................................32

Figure 11: Relative removal of oxaliplatin by activated carbon, chitosan and biochar at



............................................................................................34


and CPC by PMO-SH and PMO-SH(50%) in Milli-Q

water at pH 6 and L/S=1000, after 24 h mixing and centrifugation at 2500 rpm for 10

min (mean + standard deviation, n=3). The initial Pt concentration was 100 µg L-1

.......36

VII

Figure 13: Adsorption isotherms for adsorption of [PtCl6]2-

in synthetic urine on activated

carbon (upper left), chitosan (upper right) and biochar (bottom) (mean + standard

deviation, n=3)..................................................................................................................38

Figure 14: Adsorption isotherms for adsorption of cisplatin in synthetic urine on activated

carbon (upper left), chitosan (upper right) and biochar (bottom) (mean + standard

deviation, n=3)..................................................................................................................39

Figure15: Adsorption isotherms for the adsorption of carboplatin in synthetic urine on

activated carbon (upper left), chitosan (upper right), biochar (bottom left) (mean +

standard deviation, n=3)....................................................................................................40

Figure 16: Adsorption isotherms for the adsorption of oxaliplatin in synthetic urine on

activated carbon (upper left), chitosan (upper right), biochar (bottom) (mean + standard

deviation, n=3)..................................................................................................................41

Figure 17: Relative remaining Pt concentrations in the percolate following percolation of

synthetic urine containing [PtCl6]2-

through a column containing different combination of

biomaterials as function of eluate volume (preconditioned with 0.5 M HCl and Milli-Q

water, bed volume = 10 mL, initial Pt concentrations 100 µg L-1

)...................................42

Figure 18: Relative remaining concentrations of oxaliplatin in synthetic urine by percolating

through a filter contained of chitosan and activated carbon (blue) and activated carbon

(red) as function of eluate volume (column preconditioned with 0.5 M HCl and Milli-Q

water), flow rate and bed volume for dual bed column was 26 mL min-1

and 30 mL,

respectively while for single bed column 68 mL min-1

and 42 mL, respectively. Initial Pt


............................................................................................43

Figure 19: Relative remaining concentration of Pt in batch adsorption study with synthetic

urine containing [PtCl6]2-

and using activated carbon and chitosan (50-50) as adsorbent at

pH 7, after 24h shaking, with initial Pt concentrations of 100 µg L-1

) (mean + standard

deviation, n=3)..................................................................................................................45



through a column containing activated carbon and

chitosan as function of eluate volume (column preconditioned with 0.5 M HCl and

MilliQ-water, flow rate = 26 mL min-1

, bed volume = 30 mL & initial Pt concentration

100 µg L-1

).........................................................................................................................45

Figure 21: Relative remaining concentrations of [PtCl6]2-

in both synthetic urine (blue) and

Milli-Q water (red) following percolation through a filter contained of chitosan and

activated carbon (50:50) as function of eluate volume (column preconditioned with 0.5

VIII

M HCl and Milli-Q water), flow rate = 26 mL min-1

, bed volume = 30 mL and initial Pt

concentrations of 100µg L-1

..............................................................................................47


in synthetic urine following

percolation through a non-optimized (circular dot line) and optimized (rectangular dot

line) filter contained of chitosan and activated carbon (50:50), as function of eluate

volume (column preconditioned with 0.5 M HCl and Milli-Q water) flow rate and bed

volume for non optimized was 7 mL min-1

and 10 mL, respectively, while 26 mL min-1

and 30 mL for optimized column, respectively. Initial Pt concentration was 100µg L-

1..........................................................................................................................................48



& CPC through a column of activated carbon and

chitosan as function of eluate volume (column preconditioned with 0.5 M HCl and Milli-

Q water, flow rate = 26 mL min-1

, bed volume = 30 mL and initial Pt concentration of

100 µg L-1

).........................................................................................................................49

Figure 24: Ratio of relative remaining concentration (Ce) to the concentration of the control

(Co) during percolation of synthetic urine containing [PtCl6]2-

, cisplatin, carboplatin and

oxaliplatin through a column of activated carbon and chitosan as function of eluate

volume (column preconditioned with 0.5 M HCl and Milli-Q water, flow rate = 26 mL

min-1

, bed volume = 30 mL and initial Pt concentration of 100 µg L-1

)...........................50

IX

List of Tables

Table Page

Table 1- Global platinum supply and demand from the year 2008 to 2012 in metric ton per

year......................................................................................................................................5

Table 2: limiting toxicity and clinical status of commonly used CPC.......................................8

Table 3: Platinum emission by hospitals from Germany, Austria and Netherlands................10

Table 4: Natural and modified biomaterials previously tested for platinum removal..............15

Table 5: Initial concentrations of [PtCl6]2-

and CPC used in adsorption tests to construct

isotherms (n = 3)...............................................................................................................21

Table 6: Conditions monitored by ICP-MS.............................................................................25

Table 7: Data generated by ANOVA test after comparing different Pt species sorption on

biomaterials.......................................................................................................................27

Table 8: Data generated by ANOVA test to tudy differences in sorption effectiveness of

[PtCl6]2-

between different pH levels for different biomaterials (activated carbon, chitosan

and biochar).......................................................................................................................30

Table 9: Data generated by ANOVA test to study differences in sorption effectiveness of

cisplatin between different pH levels

for different biomaterials (activated carbon,

chitosan and biochar)........................................................................................................31


carboplatin between different pH levels for different biomaterials (activated carbon,



oxaliplatin between different pH levels for different biomaterials (activated carbon,


Table 12: Relative removal (%) of platinum species using different biomaterials at pH 7.....34

Table 13: Data generated by Mann-Whitney U test to study differences in sorption effectiveness

of [PtCl6]2-

by PMO-SH and PMO-SH(50%).......................................................................35

X

Table 14: Data generated by unpaired t-test to study differences in sorption effectiveness of

cisplatin, carboplatin and oxaliplatin by PMO-SH and PMO-SH(50%)..............................35

Table 15: Sorption isotherm parameters..................................................................................37

Table 16: Data generated by Mann-Whitney U Test to compare the single and dual bed

column use for sorption of oxaliplatin in a continous flow system...................................43

Table 17: Data generated by unpaired t-test after comparing the single and dual bed column

use for sorption of cisplatin in a continuous flow system.................................................44

Table 18: Data generated by Mann-Whitney U Test to compare sorption of [PtCl6]2-

contained

in synthetic urine and Milli-Q water by a column packed with activated carbon and

chitosan..............................................................................................................................46

Table 19: Statistical data generated by Mann-Whitney U Test to compare [PtCl6]2-

sorption

from synthetic urine between an optimized and a non-optimized column packed with

activated carbon and chitosan...........................................................................................48

Table 20: Statistical data generated by ANOVA test to compare sorption of [PtCl6]2-

,

cisplatin, carboplatin and oxaliplatin from synthetic urine on

the optimized column

packed with activated carbon and chitosan.......................................................................51

XI

Abstract The indiscriminate discharge of cancerostatic platinum compounds from hospitals is one

important anthropogenic source of platinum in the environment. Even though, they represent

a small fraction when compared to Pt emitted from cars’ catalytic convertors, they pose a

significant and higher toxicological and carcinogenic impact. Considerable portion of the

administered anti-neoplastic chemotherapies is eliminated through cancer patient’s urine.

Moreover, Pt is a precious metal, making its recovery worth to consider. Separate collection

of patient’s urine is a good alternative to achieve a more concentrated Pt stream for recovery.

Hence, the potential application of biomaterials for sorption of cancerostatic platinum

compounds and recovery from urine was investigated. The study focused on the screening

biomaterials through batch adsorption studies and evaluates their potential use in Pt recovery

through continuous flow systems. Removal efficiencies and sorption isotherms of

biomaterials were determined for [PtCl6]2-

and cisplatin, carboplatin and oxaliplatin. The

effect of pH, initial Pt concentration, urine matrix and dosage of biomaterials on adsorption

efficiency was assessed. In addition, adsorption data were calculated using the Freundlich

isotherm model. Among the biomaterials tested, activated carbon, chitosan and biochar were

selected based on their high sorption efficiency. The highest removal (78%) of [PtCl6]2-

was

achieved by activated carbon at pH 7 and no significant difference (P-value > 0.05) between

pH 6, 7 and 8 was observed. Chitosan showed higher removal at the same pH values.

Adsorption of cisplatin was less influenced by pH than [PtCl6]2-

. Adsorption of carboplatin

was not affected by pH for all biomaterials except biochar, that showed minimal influence at

pH 9 and 11. Eventually, removal of oxaliplatin by activated carbon was found the highest

(95.0%) at pH 7. In general, the result showed that sorption decreased at higher pH values.

Better removal efficiency of biomaterials at the neutral pH (7) makes them convenient for

application at the actual pH level of human urine. Capacity was higher for activated carbon,

followed by chitosan and biochar in decreasing order except for carboplatin that showed

higher adsorption for biochar than chitosan. Pt sorption followed the Freundlich isotherm

model and showed a very nice fit with strong correlation coefficient, suggesting a multilayer

adsorption system. Adsorption efficiencies of periodic mesoporous organosilica materials

PMO-SH and PMO-SH(50%) were also tested and both of them showed high removal for all Pt

species, except carboplatin. Moreover, there was a significant difference in sorption between

the two materials (P-value < 0.05) for oxaliplatin. This difference might be attributed to the

higher number (double) of thiol functional groups found in PMO-SH. A fixed bed adsorption

XII

study was conducted by a column packed with activated carbon and chitosan. The use of a

dual bed (combined biomaterials) column had significantly higher sorption efficiency (P-

value < 0.001) than a single bed column packed with activated carbon for oxaliplatin. The

dual bed column had the highest removal for oxaliplatin, followed by carboplatin, [PtCl6]2-

and cisplatin in decreasing order. Matrix effects played a significant role on adsorption of

[PtCl6]2-

due to the presence of many competing ions in the urine matrix. Eventually,

recovery of Pt was evaluated in a desorption experiment and 91.7% of the temporarily

adsorbed Pt was recovered. In general, the use of biomaterials for removal of cancerostatic

platinum compounds is considered as an option due to their high adsorption potential and

regeneration characterstics.

XIII

Acronyms AC Activated carbon

ANOVA Analysis of variance

BTICF Bayberry tannin immobilized collagen fiber

CPC Cancerostatic Platinum Compounds

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

HSD Honest significant difference

IARC International Agency for Research on Cancer

ICP-MS Inductively coupled plasma mass spectrometry

ISO International organization for standardization

PEI Polyethylenimine

PFMs Platinum group metals

pH The negative logarism of hydrogen ion concentration

PMO Periodic Mesoporous organosilica

TFMS Thiol functionalized mesoporous silicas

TOC Total organic carbon

TWA-TLV Time-Weighted Average Threshold Limit Value

WHO World Health Organization

1

1. Introduction Platinum is considered as one of the precious metals because of its low abundance in the

earth crust (Reith et al., 2014) and inertness in chemical reactions. Naturally platinum is

present in the lithosphere at low concentration that ranges from 0.001 to 0.005 mg kg-1

.

Platinum and gold are highly valued assets and they are investment products internationally

considered as forms of currency under ISO 4217 (Das, 2010). Worldwide production of

platinum has increased progressively during the past decades due to its wide use in catalytic

convertors of engine exhaust gases. Other applications of platinum are found in chemical

synthesis and petroleum industries for dehalogenation, (de)hydrogenation, isomerisation and

oxidation reactions (WHO Regional Office for Europe, 2000).

The potential use of platinum derivates for cancer treatment has been introduced after the first

discovery by Rosenberg et al. (1965) of the inhibition of cell division on Escherchia coli

culture by a platinum electrode in TRIS buffer. Since then, many platinum complexes are

being used for treating cancer patients (Ravindra et al., 2004; WHO Regional Office for

Europe, 2000).

The wide and ever increasing application of this noble metal has elevated its emission and

concentration in the environment (Michalke, 2010). The distribution and spread of Pt to

airborne particulate, road side dust and soils is due to its intensive use in catalytic converters.

It is washed out by erosion and concentrated in sewage sludge that eventually leads to

bioaccumulation in living organisms through different pathways (Schafer & Puchelt, 1998).

In addition to its use in automobile catalysts and industry, there is a growing demand and

utilization of Pt complexes for treating lung and bladder cancer, testicular and metastatic

ovarian tumours, and tumours of the head and neck. The most widely utilized platinum-based

drugs applied for successful treatment of human malignancies are cisplatin [cis-

diaminedichloro platinum (II)], carboplatin [diamine (1,1-cyclobutanedicarboxylato)

platinum(II)] and oxaliplatin {(SP-4-2)-[(1R,2R)cyclohexanediamine-k2N,N’][(ethan-

dioato(2-)-k2O

1,O

2]-platinum(II)} (Michalke, 2010, Ravindra et al., 2004).

However, the great majority of these antineoplastic drugs that contain platinum is discharged

to hospital effluents since they are mainly excreted through urine of patients treated with

these medicine. A study conducted in five European hospitals (Austria, Belgium, Germany,

2

Italy and The Netherlands) showed that about 70% of the administered Pt (cisplatin or

carboplatin) ended up in the effluent (Kummerer et al., 1999).

Studies showed that from human pharmaceuticals, mainly antibiotics and anti-cancer drugs

are found in ng L-1

to µg L-1

concentrations in surface water and other environmental

compartments. This should mainly be attributed to low removal efficiencies of conventional

sewage treatment plants towards these compounds and their metabolites. Once released into

the environment, the pharmaceuticals may have negative impacts on the environment.

Therefore, the continuous discharge of human pharmaceuticals to the environment from

hospitals demands sincere attention (Zhang et al., 2013).

Studying its impact and fate is however complicated by the fact that speciation plays a role in

the bioavailability, biological activity and (eco) toxicity of Pt. For instance, chloro-platinum

compounds are readily soluble in water and cause damage to health, while elemental and

platinum dioxide are rather insoluble (Ljubomirova & Djingova, 2008; Ravindra et al., 2004).

One of the approaches to reduce emission of platinum from hospitals that should be

considered is the separation of effluent (urine) discharged from oncology departments from

the other waste streams. As these effluents contain the cancerostatic platinum compounds

(CPC) and their excretion products at higher concentrations, treatment and recovery

processes may become more feasible, both from a technical and an economic point of view

(Zhang et al., 2013). In addition to the development of techniques focused on removal of Pt

from wastewater and urine for environmental purposes, its recovery and reuse are also worth

to be explored as Pt is a valuable element, with prices exceeding those of gold.

Several conventional methods may be used to remove metals from waste water, which

include oxidation, reduction, precipitation, membrane separation, filtration, ion-exchange,

electrochemical treatment and adsorption. All of these methods except adsorption, are

considered as rather expensive, have low specificity towards compounds and are hardly

capable of removing trace levels of Pt from waste waters. However, the use of biomaterials

as sorbent for precious metals like platinum is gaining increasing attention since they are both

economical and environmental friendly (Fujiwara et al., 2007; Prabakaran & Arivoli, 2012).

Therefore, this study was conducted to assess the potential use of biomaterials for the

removal and recovery of CPC from urine.

3

2. Objectives

2.1 General objective

The objective of this master thesis is to determine the potential of biomaterials as sorbent for

removal of CPC from human urine.

2.2 Specific objectives

The entire study is further divided into each of the following distinct goals:

Determine potential and capacity of biomaterials for Pt sorption in a batch

adsorption study

Determine factors that affect sorption of Pt compounds by biomaterials, including

the evaluation of pH impact and matrix effects of urine on adsorption behaviour

of CPC using a continuous flow system

Compare the efficiency of biomaterials separately and in combination to remove

different Pt compounds in batch and continuous flow

Conduct a column experiment aimed at assessing the feasibility to operate the

adsorption process under continuous flow condition

4

3. Literature review

3.1 History of platinum use, its sources and release in to the environment

Platinum is a noble metal known by its silvery white, ductile and malleable nature, which is

scarcely distributed over the earth’s crust. Mostly, it is found as isotopes with atomic weights

of 194, 195, 196 and 198 and the most stable oxidation states are + II and + IV (WHO

Regional Office for Europe, 2000).

It belongs to the Platinum Group Metals (PGMs), which include palladium (Pd), ruthenium

(Ru), osmium (Os), iridium (Ir), and rhodium (Rh) and are all low abundant elements.

Platinum is known for its non-reactive refractory nature and having high boiling and melting

points, which gives it a very strong resistance against corrosion and oxidation (Reith et al.,

2014).

Although it is rare in nature, the recent demand of platinum in a versatile range of

applications has made a significant contribution to its wide spread occurrence. The major

application is in catalytic convertors in cars which were introduced in 1975 in the USA as a

response to the stringent air emission limit of the Federal Clean Air Act. Afterwards, since

1993, the catalytic convertors became compulsory also in Europe. On average, a catalytic

converter of a motor vehicle contains about 1-3 g of platinum, making the world-wide annual

emission of platinum from vehicles as high as 0.5-1.4 metric ton. Taking in to account

distances, an emission factor in the range of 65 ng km-1

y-1

to 180 ng km-1

y-1

can be

established (Ravindra et al., 2004).

Significant platinum uses are also found in ammonia oxidation, during the production of

nitric acid and for catalytic upgrading of the octane rating of gasoline. Besides these wide

applications, it is also used in glassmaking, dentistry, jewelry, prosthetics, pacemaker

electrodes and drugs for anticancer treatment (Reith et al., 2014). According to Johnson

Matthey, (2013) there were no large changes in the supply of platinum for the medical and

biomedical sector between 2008 and 2012 (Table 1).

5

Table 1- Global platinum supply and demand from the year 2008 to 2012 in metric ton per

year (Johnson Matthey, 2013)

Platinum use

Year

2008 2009 2010 2011 2012

Automobile catalyst 103.6 61.9 87.2 90.3 91.9

Chemical 11.3 8.2 12.5 13.3 12.8

Electrical 6.5 5.4 6.5 6.5 4.7

Glass 8.9 0.3 10.9 14.6 5.1

Investment 15.7 18.7 18.6 13.0 12.9

Jewellery 58.4 79.7 68.6 70.2 78.8

Medical & biomedical 6.9 7.1 6.5 6.5 6.7

Petroleum 6.8 6.0 4.8 6.0 5.7

Other 8.2 5.4 8.5 9.1 9.6

Total supply 168.4 170.8 171.5 183.8 159.9

Total gross demand 226.5 192.6 224.1 229.5 228.1

Among the above mentioned major platinum uses, only automobile catalyst and jewellery

have managed to recycle a significant amount of platinum, while sectors like the medical and

biomedical sector did not have any means to recover the precious metal (Johnson Matthey,

2013).

According to the study done by Nischwitz et al. (2004) platinum production has increased

from 127 tons in 1971 to 152 tons in 1995 and it is expected to rise further due to its current

demand in the different applications described above. In the last couple of decades, the

consumption of CPC has increased reasonably (WHO Regional Office for Europe, 2000).

Platinum is found in different environmental samples in varying concentrations. It has been

detected in soil dust, water, air-born particles, sewage sludge, hospital effluent, incinerated

ash, etc. According to Jackson et al. (2007) the platinum released from medical facilities in

Sheffield has resulted in a concentration of 36 ppb for the urban sewage (Fig.1). Moreover,

due to the administration of antineoplastic drugs in cancer patients, platinum may also be

found in biological samples like blood and urine in high concentrations. A research done by

Lanjwani et al. (2006) has shown that the excretion amount of Pt through urine has increased

6

over time. Pt content on the urine samples collected 8 h after the infusion of cisplatin was 43

ng mL-1

and this value increased to 97 ng mL-1

after 24 h. Another study conducted by

Kummerer et al. (1999), also described that 31-85% of cisplatin is excreted through urine

within 51 days. This shows that CPC excretion continued even after the cancer patients are

discharged from hospitals. Therefore, treatment and recovery of Pt shouldn’t be limited to the

hospital setup only. Patients need to be tracked and recovery systems have to consider the

diffused source as well, which are the households (Kummerer et al., 1999).

Figure 1: Concentrations of platinum and palladium in parts-per-billion for different

environmental samples in Sheffield, UK. And show the maximum values measured by

Jackson et al. (2007).

Determination of platinum in biological samples was not reliable before the 1980s since the

concentrations in the sample were found below the detection limit of the instruments

available at that time (WHO Regional Office for Europe, 2000). Nowadays, the presence of

Pt in all environmental compartments can be analysed by the availability of advanced

instrument like inductively coupled plasma-mass spectroscopy (ICP-MS), which can

determine to the ng L-1

level. Hence, advances in science and technology help to evaluate the

presence and emission of Pt to the environment across the globe.

7

3.2 Cancerostatic platinum compounds (CPC)

The speciation of platinum received a lot of attention when CPC became used extensively in

biological and clinical areas. During cancer treatment using CPC, the kinetic effects in vivo

and presence in wastewater is entirely determined by the speciation, indicating the impact of

speciation on the activeness of the Pt compounds (Michalke, 2010).

The application of platinum compounds for clinical purposes is characterized by presence of

very high concentrations in the blood of patients, up to the low mg Pt L-1

range (Ljubomirova

and Djingova, 2008). However, its concentration in the environmental samples is rather low.

A study done by Ravindra et al. (2004) revealed that the average daily concentration of Pt in

hospital effluents was < 10 ng Pt L-1

for hospitals sampled from Belgium and Italy while

3,500 ng Pt L-1

for Germany and Austrian hospital. Whereas, the platinum emissions from

catalytic convertors, at a simulated speed of 100 km h-1

, in the exhaust gas was 17 ng m-3

.

The Pt found in the hospital effluent is much higher than in the exhaust gas. Moreover, WHO

task group on environmental health criteria for platinum had decided that Pt containing

exhaust emission especially from monolithic three-way catalyst most likely do not pose any

health risk to the general population (WHO Regional Office for Europe, 2000).

3.2.1 Platinum containing chemotherapy

Rosenberg et al. (1965) first discovered the property of platinum to inhibit cell division.

Afterwards, platinum containing coordination complexes were developed, with cisplatin (cis-

diaminedichlororplatinum(II)) being the first cancer treating drug in the 1970s (Barefoot,

2001). It was once called as “penicillin of cancer drugs” and considered as the parent

compound for this class of agents (Michalke, 2010; Desoize and Madoulet, 2002; Lippert,

2013). Since then many more Pt containing compounds were developed and commercially

produced, especially in the last couple of decades (Michalke, 2010). Platinum based

chemotherapies are now considered as the most effective drugs against many types of

malignancies (Zhang et al., 2013).

However, platinum based chemotherapies are only effective for some kinds of cancers and

cause severe side effects. Nephrotoxicity is the main dose limiting side effect following

administration of the drug. Cisplatin, which is more toxic than other platinum based drugs,

8

has a higher binding affinity towards proteins. This property affects its efficiency, level of

toxicity and time and amount of CPC excreted through urine (Bareffoot, 2001; Michalke,

2010). The second generation drugs, like carboplatin, have managed to reduce the side effects

to some extent. The most common platinum containing cancer treating drugs with their

clinical status and toxicity are presented in Table 2.

The most common cancerostatic platinum compounds of the second generation are

carboplatin (cis-diammine 1,1-cyclobutanedicarboxylatoplatinum) and oxaliplatin ([(1R,2R)-

1,2-cyclohexanediamine- N,NV]oxalate(2-)-O,OV-platinum) (Fig. 2). Cisplatin and

carboplatin are used to treat aggressive cancer types like testicular tumours, ovarian

carcinomas, bladder tumours, and tumours of the head and neck, while oxaliplatin is

approved and preferred for colorectal cancer treatment besides its use for other types of

cancer (Lenz et al., 2005; Michalke, 2010; Barefoot, 2001). Moreover, some other platinum

containing drugs used for cancer therapy include lobaplatin, nedaplatin and heptaplatin,

applied regionally in China, Japan and South Korea respectively (Michalke, 2010).

Cisplatin Carboplatin Oxaliplatin

Figure 2: Structural formula of three platinum derivatives (Desoize and Madoulet, 2002)

Table 2: limiting toxicity and clinical status of commonly used CPC (Barefoot, 2001 and

Boulikas et al., 2007)

Drug Main limiting toxicity Clinical status

Cisplatin Nephrotoxicity, myelosuppression Worldwide approval

Carboplatin Myelosuppression Worldwide approval

Oxaliplatin Neuropathy Worldwide approval

Lobaplatin Thrombocytopenia Approved in China;

Phase II clinical trials

Nedaplatin Myelosuppression Approved in Japan

JM216 Myelosuppression Phase III clinical trails

9

3.2.2 Mechanism of action of CPC

In the cell, it is believed that cisplatin go through hydrolytic activation and react with DNA,

which is a nuclear target molecule (Lanjwani et al., 2006). Inhibition of cancer cell growth is

executed by CPC that form different kinds of adducts including transcription, block

replication and induce cell death (Boulikas et al., 2007). The loss of chlorido, dicarboxylato

and oxalato ligands in cisplatin, carboplatin and oxaloplatin respectively, leads to

coordination with DNA nucleotides as the main target. Among the DNA nucleotides, the N7

intrastrand cross-linking with two adjacent guanine units is the principal adduct that creates

serious distortion on the DNA double-strand structure that consequently results in apoptosis

and cell death (Lippert, 2013).

3.2.3 Exposure route

The major pathway for platinum exposure is via food with an average intake rate of 1.44 μg

day-1

for adults and reasonable difference between males and females. The two main groups

in which elevated platinum exposure is observed are occupational exposed workers and

patients treated with platinum based chemotherapeutic agents intravenously. Although the

time-weighted average threshold limit value (TWA-TLV) was set to 2 μg m-3

for soluble

platinum salts and 1 mg m-3

for platinum metal. Early researches conducted on

occupationally exposed workers showed that work place platinum level was found in a range

of 0.9-1,700 μg m-3

(WHO Regional Office for Europe, 2000). Moreover, a study done by

Venitt et al. (1984), showed that the urine of cancer patients treated with cisplain was found

with average Pt concentration of 7 mg L-1

that suggests any contact with it might be very

hazardous.

3.2.4 Environmental and human health impact of CPC

Platinum group metals exist in all environmental compartments. People exposed to them in

various ways. For instance, studies have shown that workers in precious metal refineries were

found with elevated concentrations of platinum in their body. Exposure to the metallic

platinum, which is inert to biological transformation, shows only mild toxicity, while some

platinum salts, like hexachloroplatinate and tetrachloroplatinite are the most dangerous

allergens and sensitizers. Therefore, exposure to platinum group elements is often correlated

to health problems like nausea, asthma dermatitis, increased spontaneous abortion and other

severe health problems (Ravindra et al., 2004).

10

The coordination complexs are also considered to have elevated biochemical activity. The

cytotoxicity and mutagenicity of cisplatin is considered to pose potential health problems to

the public when chronically exposure to even low amounts of Pt. The International Agency

for Research on Cancer (IARC) has included cisplatin in group 2A, which is levelled as

‘probably cancerogenic to humans’. Hence, the small concentration found in hospital

effluent or municipal sewage should be seriously considered as a potential public health

problem (Ljubomirova and Djingova, 2008; Lenz et al., 2005).

3.2.5 Contribution of hospitals towards platinum concentrations in municipal sewage

Hospitals are one of the major emission sources that should be taken into consideration as far

Pt emission is concerned, since there is a direct discharge to the environment and the emitted

compounds are considered to have potential environmental and public health effects. A study

done by Kummerer et al. (1999), on five European hospitals indicated that hospitals are

releasing a considerable amount of platinum. In Germany, from the total of 100 - 400 kg of Pt

input to the sewage system, 14.2 kg is contributed by hospitals (Table 3). The same study

indicated that after administration of cisplatin and carboplatin, the majority of the ingested

drugs is excreted through urine. For instance, 50-75% of the carboplatin is excreted within 24

hours.

Table 3: Platinum emission by hospitals from Germany, Austria and The Netherlands

(Kummerer et al., 1999)

Germany Austria The Netherlands

Year 1996 1996 1996

Total hospital beds studied 1,887 2,514 857

Pt per bed and year (mg) 130.4 58.7 22.3

Total emission by hospitals (kg) 14.2 NA NA

NA, not available;

11

3.3 Options for platinum removal and recovery from wastewater

The usual approach of co-treating hospital wastewater with urban wastewater in municipal

wastewater treatment facilities is insufficient to remove Pt compounds and other

pharmaceuticals because of their specific nature and low concentration. Practically no

separation of wastes is done, especially from hospitals that contain emerging contaminants

which become diluted and find their away to the environment without proper treatment. For

example, the average concentration of platinum in hospital effluent and urban waste water

was found to be 13 µg L-1

and 0.155 µg L-1

respectively (Verlicchi et al., 2010). Especially,

this low concentration makes CPC difficult to be removed. Besides this, a wide range of other

compounds also occur in mixed effluents, which may affect their final fate during the

treatment (MWWT) (Verlicchi et al., 2010).

Although various types of treatment methods have previously been are suggested, biosorption

draws increasing attention over the conventional methods due to its low cost, high efficiency,

minimum generation of chemical and biological sludge, and possibility of regenerating the

biosorbent and recovering precious metals for reuse instead of using virgin materials.

Therefore, taking into account their rare nature, recovery of these metals from aqueous

stream is rational (Das, 2010; Sud et al., 2008).

3.3.1 Biosorption

Biosorption is described as the removal of compounds, metal ions, etc. using inactive or non-

living biomass (materials of biological origin) as a result of the existing interaction between

them. Metabolically inactive (dead) and living materials are capable of removing metals due

to the different functional groups found in their cells, which provide them high efficiency

(Farooq et al., 2010). A study conducted by Sud et al. (2008) has indicated that there are

many materials with high potential for metal sorption originating from cellulosic, agricultural

products. The common functional groups found in these agricultural materials include:

phenolic, carbonyl, acetamido, alcoholic, amido, amino, sulphydryl groups, etc. which have

high affinity towards metals to form metal complexes or chelates.

12

3.3.2 Biomaterials used for sorption of platinum

Many studies focus on the potential application of biomaterials for removal of micro

pollutants and toxic elements from different aqueous streams. Efficiencies of some of the

materials that have been tested already were found impressive. Moreover, biomaterials are

locally available, biodegradable and, environmentally friendly. Recovery of the precious

metal is simple by regeneration, and the biomaterial can often also be reused (Fujiwara et al.,

2007; Zhou et al., 2009).

Basically, all kinds of biological materials have some sort of natural tendency for adsorption

of organic and inorganic pollutants and their biosorption potential differs among themselves.

Biomaterials can be either organic or inorganic in nature and found in different forms, each

influencing physico-chemical process like adsorption, ion exchange, precipitation and surface

complexation (Gadd, 2009). The challenge is to select the material that can remove pollutants

to an acceptable level (Fomina & Gadd, 2014).

Materials that can be used for metal recovery through adsorption include activated carbon,

natural and modified chitosan, biochar, clays, metal oxides, silica and zeolite, coffee husk,

saw dust, biological organisms, etc. (Sharififard et al., 2012; Dutta et al., 2004; Zhou et al.,

2009; Chen et al, 2014).

Chitosan

Chitosan is partially acetylated glucosamine biopolymer and a primary product of

deacetylation of chitin (Zhou et al., 2009). Chitin is the most abundant natural polysaccharide

on earth next to cellulose and it is a whitish hard nitrogenous polysaccharide, which is found

in the exoskeleton of invertebrates (Fig.3) (Dutta et al., 2004; Chang et al., 2012).

Figure 3: Structure of chitosan (Islam et al., 2011)

13

Chitosan is a renewable polymer, which is known by its biodegradability, non-toxicity,

adsorption capability and antibacterial nature that selects for a wide range of applications

including biomedical, food and textile. Different studies showed that chitosan is an excellent

adsorbent for metal removal. It has a high nitrogen content, which helps it to take up

numerous metal ions through different means depending on the metal ion available and pH of

the solution. The amine and two hydroxyl groups found in each glucosamine unit are reactive

sites, which make chitosan suitable for chemical modification (Dutta et al., 2004; Ramesh et

al., 2008; Sharififard et al., 2012).

In general, the processes involved in pollutant removal when using chitosan and chitin

include adsorption (28%), coagulation (4%), precipitation (7%), filtration (4%), flocculation

(3%), flotation (1%), and membrane filtration (53%). Chitosan also serves for treatment of

waste water that contains heavy metals, radionuclides and dyes when sufficient amino and

hydroxyl functional groups are present (Wang & Chen, 2014).

Chitosan can be applied in its original form or modified through crosslinking, blending, graft

polymerization or internal hydrogen bonding. Modification is important because chitosan in

its original form lacks mechanical strength and becomes soluble in acidic media. Besides that

it has no specific selectivity for certain heavy metals and has very limited sorption capacity

for complex mixtures of waste water. However, chitosan with high level of amine and

hydroxyl functional groups show high sorption capacity. This is especially the case for

transition metals and, to a lesser extent, for alkaline or alkaline earth metals. Therefore,

modification improves its stability in acid media and increases the sorption capacity (Wang &

Chen, 2014).

Activated carbon

Use of activated carbon for metal recovery and removal is an old and well developed

technique. Adsorption using activated carbon is highly effective and a very suitable method

for large volumes of highly diluted solutions. Especially, the high internal surface in a range

of 500 m2

g-1

to 1,500 m2

g-1

makes it suitable for adsorption. It can be found in two different

forms, powdered activated carbon (PAC) and granular activated carbon (GAC) depending on

the particle size (Lenntech, 1998). Activated carbon is considered as an ideal adsorbent as far

14

as precious metal recovery is concerned, due to very high adsorption capacity, high rate of

adsorption and good resistance towards mechanical abrasion (Sharififard et al., 2012).

For instance, gold recovery using activated carbon is the most dominant process after

leaching by cyanide in aerated alkaline slurries, whereas granular activated carbon was used

to remove precious metals like platinum, palladium and gold with great preference over base

metals, which include nickel and copper from low-grade ores (Mpinga et al., 2014). Another

study conducted by Modin et al. (2011) indicated that activated carbon was able to remove

metals like Co, Cr, Fe, Mn and Ni from land fill leachate.

Biochar

Biochar is a porous substance, which is quite similar to charcoal in appearance and produced

by pyrolysis of feedstock under oxygen limited conditions. Pyrolysis can be performed by

utilizing different inputs like lingo cellulose feed stocks, agricultural residues and biorefinery

wastes (Kołodynska et al., 2012).

Although biochar has a relatively lower specific area and micropore volume than commercial

activated carbon (AC), its adsorption capacity is similar to that of AC. Its chemical and

physical properties are based on the feedstock source and condition of pyrolysis. Temperature

is one of the crucial conditions for pyrolysis and has a critical role on the nature of biochar. If

the temperature is very high, the loss in surface functional groups and carbon will be

significant. According to Chen et al. (2014) the optimum temperature for pyrolysis of biochar

was found to be 900 0C for efficient energy (H2 and syngas) recuperation and metal recovery

by having efficient microstructures.

Other biomaterials

Any agricultural waste, which is readily available and demands very minimal resources for

conversion processes, is also appropriate for waste water treatment (Oliveira et al., 2008).

Other options are coffee husk and saw dust, which are cellulose based materials that contains

tannin and lignin based organic compound (Naiya et al., 2009). Many researches previously

applied biomaterials for removal of platinum. Some of their studies are presented in Table 4.

15

Table 4: Natural and modified biomaterials previously tested for platinum removal

Adsorbent pH Amount of metal

sorbed

Initial

concentration

L/S Reference

Activated carbon 2 45.5 mg g-1

50-300 mg L-1

1:166 Sharififard et

al., 2012

Saccharomyces

cerevisiae & green algae

1.6-1.8 Yeast 0.8 µg g-1

of biomass

(algae 71.4+2.9%

removal efficiency)

75 µg L-1

1:133

(column

test)

Godlewska-

Zylkiewicz,

2003

Polyethylenimine (PEI)-

modified biomass

_ 108.8 mg g-1

_ 1:10 Won et al,

2010

l-lysine modified cross

linked chitosan resin

1 129.26 mg g-1

10-400 mg L-1

- Fujiwara et al.,

2007

Glycine modified

crosslinked chitosan

resin

2 122.4 mg g-1

50-500 mg L-1

1:300 Ramesh et al.,

2008

Thiourea-modified

chitosan Microspheres

2 129.9 mg g-1

10-400 mg L-1

- Zhou et al.,

2009

bio-polymer modified

activated carbon

2 52.63 mg g-1

50-300 mg L-1

1:166 Sharififard et

al., 2012

Bayberry tannin

immobilized

collagen fiber (BTICF)

membrane

- Below detection until

700 mL in column

test

58.3 mg L-1

Column

test

Das, 2010

Thiocarbamoyl chitosan

(TC-chitosan) derivatives

_ 1.24 mmol g-1

20-300 mg L-1

1:2000 Bratskaya et

al., 2011

3.4 Factors affecting biosorption

Besides the physico-chemical nature of the sorbate and sorbent, there are different factors that

affect biosorption. These critical factors include: solution of pH, ionic strength, initial

pollutant concentration, presence of other pollutants that compete for sorption, the nature of

the biosorbent and available binding sites, temperature and agitation speed (Fomina and

Gadd, 2014). In particular, pH is a very important parameter that affects speciation of metal

in a solution by playing a role through redox reaction, hydrolysis and complexation during

metal recovery. Hence, the pH can be modified to enhance maximum biosorption (Das,

2010).

16

3.5 Sorption models and isotherms

The adsorption process is modelled and quantified by adsorption isotherms. The adsorption

isotherm is the curve describing the distribution of molecules between the liquid phase and

the solid phase after reaching equilibrium conditions. Equilibrium sorption isotherms help to

determine and predict the capacity of adsorbents and their performance. Even though several

theories have been forwarded to depict mechanisms of adsorption, the two most common and

widely used models are the Langmuir and Freundlich models (Okeola & Odebunmi, 2010;

Ghasemi et al., 2014).

The Langmuir isotherm model has assumptions that include the adsorbent surface being

homogenous, atoms or ions following a mono-layer adsorption process and the absence of

interaction between adsorbates on the sorbent surface. The Langmuir equation is represented

as:

eq

eqeq

bC

bCqq

1

max

Where, qeq is the total amount of metal sorbed at equilibrium state (mg g-1

), qmax is the

monolayer sorption capacity (mg g-1

), b is Langmuir constant, Ceq holds for the metal

concentration in the solution at equilibrium (Farooq et al., 2010). The linear form of the

equation can also be presented as follows.

maxmax

1

q

C

bqQ

C eq

eq

eq

Where, Ceq (mg L−1

) is the equilibrium concentration of metal ions, Qeq (mg g−1

) is the

amount of metal ions adsorbent at equilibrium state, qmax (mg g−1

) and b (L mg−1

) are

Langmuir constants, which refers to the adsorption capacity and adsorption energy,

respectively. Parameters of Langmuir isotherm (qmax and b) derived from slope and intercept

of the plot of Ceq/Qeq versus Ceq. The correlation coefficient (R2) determines applicability of

the model (Ghasemi et al., 2014; Prabakaran and Arivoli, 2012).

17

The Freundlich model considers, unlike the Langmuir model, reversible adsorption. The

formation of mono layer adsorption is moreover not mandatory. According to Prabakaran and

Arivoli (2012) the Freundlich isotherm model is expressed as follows.

/n1

eqfCk eqq

Where, Kf and n are Freundlich coefficients that refer to the adsorption capacity and

adsorption intensity of the adsorbent respectively. n is an indicator for favorable condition for

adsorption i.e. if n > 1, there are good conditions for the adsorption process to take place

(Ghasemi et al., 2014).

18

4. Materials and methods

4.1 Introduction

The study included the use of different biomaterials. The biomaterials considered were

powdered and granular activated carbon, biochar, chitosan, grass, coffee husk and saw dust.

Based on their efficiency and characteristics, only the first three were selected for further

testing and optimization. Moreover, a thiol containing Periodic Mesoporous Organosilica,

(PMO-SH and PMO-SH (50%)) with different proportions of the functional group, which is not

a biomaterial, was also tested for its platinum sorption efficiency. In general, the selected

materials and their combinations were tested for sorption efficiency and to establish

isotherms using batch adsorption tests and for feasibility to operate the adsorption process

under continuous flow conditions using column test. The PMO material was only tested in

batch method since it was available only in small amounts and fine powdered form, which

could not be used in a column test.

4.2 Variables

This study has considered several dependent and independent variables affecting the sorption

sorption. These were the initial sorbate concentration, pH, total organic carbon (TOC),

conductivity and chloride concentration. They were measured and /or controlled to determine

the key factors that affect sorption and to calculate efficiency, sorption isotherm and removal

in batch and continuous flow system.

4.3 Materials

Medium molecular weight chitosan (75% to 85% deacetylated) was purchased from Sigma-

Aldrich (St. Louis, MO, US). Activated carbon from Merck & Co. Rahway, N.J (White

House Station, NJ, US) was used for mixing with other adsorbents and as reference

adsorbent. Powder and granular activated carbon were used, having a size of 0.15 mm and 2

mm diameter respectively. The biochar used was prepared from beech biomass with a size of

2 mm. PMO-SH was prepared by the Department of inorganic and physical chemistry of

Ghent University.

19

All reagents used were reagent grade. Stock solutions and standards were prepared using

fresh Milli-Q water. For the preparation of synthetic urine distilled water was used. The pH

of the aqueous medium was adjusted by ultra pure HCl and ammonia solution (Chem-Lab

NV, Zedelgem, Belgium). Preconditioning of the filter column packed with biomaterials right

before the sorption test was done by using similar quality 0.5M HCl and subsequent washing

with Milli-Q water. Filter media were regenerated was also done using pure grade HNO3

diluted to 0.5M.

Platinum solutions used for both batch and column tests include: [PtCl6]2-

, cisplatin,

carboplatin and oxaliplatin. Desired concentrations of [PtCl6]2-

for sorption and calibration

curve of the instrument were prepared from 1000 mg Pt L-1

stock solution (Chem-Lab NV,

Zedelgem, Belgium) while CPC (cisplatin, carboplatin and oxaliplatin) solutions were

prepared by weighing powders purchased from Sigma-Aldrich (St. Louis, MO, US) and

dissolving them in Milli-Q water.

4.3.1 Synthetic urine

Human urine was synthetically prepared. The synthetic urine contained 17 g L

-1 sodium

chloride, 49 g L-1

urea, 7.6 g L-1

potassium chloride, 2.06 g L-1

citric acid, 2.36 g L-1

of

potassium phosphate, 0.68 g L-1

of ascorbic acid, 1.28 g L-1

sodium hydroxide, 0.94 g L-1

sodium bicarbonate, and 2.8 g L-1

creatinine. To prepare it, these products were added to 500

mL deionised water to the desired concentration and 0.56 mL concentrated sulphuric acid

was also added. Then the solution was stirred for 1 h at 650 rpm and stored at –4 °C until

further use. The synthetic urine was reheated to room temperature to adjust it to its natural

condition (25 oC) when used.

4.4 Methods

4.4.1 Batch adsorption study

In each batch test 0.1 g of the biomaterial was weighed and put in to a 50 mL centrifuge tube

containing 19.8 mL of synthetic urine spiked with 0.2 mL 10 mg L-1

platinum solution. This

resulted in a final volume of 20 mL with a concentration of 100 µg Pt L-1

and a L/S ratio of

200. The initial pH (Orion Star A211 instrument) of synthetic urine was 6 and it was further

20

adjusted in the range of 5 to 11 with an interval of 1 for [PtCl6]2-

and 2 for CPC by using HCl

and ammonia solution.

Subsequently, the tubes were shaken on a mechanical shaking plate for 24 hours to reach

equilibrium concentrations. Solid-liquid phase separation was done by filtration over a

Millipore filter paper (0.45 μm pore size) and 5 mL of the filtrate was mixed with 3 mL HCl

and 1 mL HNO3 in open vials prior to microwave digestion (CEM, Matthews, NC, US). The

digestion temperature was raised to 55 oC 10 min, then to 75

oC for another 10 min and

finally to 110 oC for 40 min to entirely degrade the matrix. Eventually, the digested samples

were cooled down and Milli-Q water was added to reach the final volume of 10 mL. After

dilution with 10 µg L-1

indium as internal standard, all samples were analysed using ICP-MS

(PerkinElmer SCIEX, Waltham, MA, US).

The sorption test using mixed biomaterials was done by taking a 50-50 mix ration from each

biomaterial. The adsorbent dose used for the mixture was 0.05 g from each biomaterial that

makes the final dose 0.1 g, which is equal to the treatment dose when pure biomaterials. The

procedure for testing mixed biomaterials was similar to the procedure described for testing

pure treatment. The control groups, containing only the platinum solution, were used to take

into account sorption that may occur on the surface of the vials. All tests were done in

triplicate and platinum removal was calculated as follows.

100*)((%)Re0

0

C

CCremovallative

e

Where: C0 is the concentration in the control group and Ce is the Pt concentration after

adsorption (Ramesh et al., 2008).

Moreover, it can be also presented as relative remaining concentration

100*)((%)ion concentrat remainingRe0C

Clative

e

Where: C0 is the concentration in the control group and Ce is Pt concentration after

adsorption.

A batch adsorption study was also used to test the platinum sorption potential of thiol

containing Periodic Mesoporous Organosilica (PMO-SH) and PMO-SH(50%) (the thiol

functional group is half of the first one). PMO-SH contains CH2-CH2-SH groups in-to a

21

framework that gave a sulphur content of 13.2%. The amount of sulphur in each vial (10 mg

of PMO-SH) was found to be 0.0427 mmol. PMO-SH(50%) incorporated unsubstituted -CH2-

CH2- groups in combination with the CH2-C2H-SH in a ratio of 50:50. This results in a

sulphur content of 6.17%. Each vial that holds 10 mg of PMO-SH(50%) contains 0.0192 mmol

S during the batch adsorption test.

An amount of 0.01 g of PMO-SH / PMO-SH(50%) was mixed with 10 mL of Pt solution that

results in a L/S ratio of 1000. Pt species were added to each vial yielding an initial

concentration of 100 µg L-1

. The samples were shaken for 24 hours and separation of liquid

and solid phase was established by centrifuging for 10 min at 2,500 rpm (Eppendorf 5804 R,

Hamburg, Germany). 5 mL aliquot was then taken, digested with acids and analysed with

ICP-MS following the same procedure as mentioned above. Sorption efficiency was also

calculated by the formula described above. Eventually, regeneration of the PMO-SH and

PMO-SH(50%) was done by using 0.5 M HNO3 and de-ionized water.

4.4.1.1 Concentration dependency and construction of isotherms

The effect of initial concentration on sorption was studied by an adsorption equilibrium

experiment, following the same procedure as described for the batch adsorption test above.

However, different initial Pt concentrations were used. They varied in a range from 1 µg L-1

to 10 mg L-1

and are presented in Table 5 below.

Table 5: Initial concentrations of [PtCl6]2-

and CPC used in adsorption tests to construct

isotherms (n = 3)

Pt Species [PtCl6]2-

Cisplatin Carboplatin Oxaliplatin

Concentration

(µg L-1

)

1 1 1 1

5 - 20 -

20 20 52.5 20

100 100 100 100

200 200 200 200

500 500 500 500

1,000

10,000

22

Adsorption capacity of the biomaterials was calculated as follows.

Vm

CCq

ee

0

Where, qe is the amount of metal ions sorbed to the biomaterial (µg g-1

), C0 is the initial

concentration (control group) and Ce the equilibrium metal ion concentrations (µg L-1

), m is

the dry weight of the biomass used (g) and V is the volume (L). The sorption capacity allows

to compare the different biomaterials since it takes in-to account the weight of the biomaterial

used (Farooq et al., 2010).

Langmuir and Freundlich models were used to evaluate the adsorption data.

The Langmuir equation is expressed as:

eq

eqeq

bC

bCqq

1

max

Where, qeq is the total amount of metal sorbed at equilibrium (mg g-1

), qmax is the monolayer

sorption capacity (mg g-1

), Ceq stands for the metal concentration in the solution at

equilibrium and b is Langmuir constant (Farooq et al., 2010).

The Freundlich isotherm model is presented by the formula

/n1eqfCk eqq

Kf and n corresponds to Freundlich constants representing the adsorption capacity and the

adsorption intensity, respectively.

Freundlich isotherm model was constructed by using sigmaplot version 12 statistical software

packages. While the linear form of Langmuir was calculated on Microsoft Excel 2007.

4.4.2 Fixed bed adsorption study (column test experimental set up)

Columns packed with individual or mixed biomaterials were installed to make a continuous

flow system. The weight of biomaterials needed for the column was calculated from the

capacity test Vq

CCm

e

e

0

23

Where, m is the dry weight of the biomass required (g), qe is the amount of metal sorbed at

equilibrium concentration of 100 µg Pt L-1

, C0 is the initial concentration (control group) and

Ce the equilibrium metal ion concentrations (µg L-1

), V is the volume (L).

The calculated adsorbent dose was doubled considering the short contact time of Pt solution

with the adsorbent while percolating through the column. Besides testing the three adsorbents

individually, mixtures of them also evaluated. The mixtures were activated carbon with

chitosan, activated carbon with biochar and eventually chitosan with biochar. Among these

combinations, the mixture with the highest removal was considered for optimization. The

column was preconditioned with 0.5 M HCl and washed by Milli-Q water prior to the

sorption test (Fig. 4).

Figure 4: Columns containing biomaterials for continuous adsorption study

The pH of the synthetic urine was adjusted to 9 and 250 mL platinum solution was flushed

through the packed column. Leachate was collected continuously by 25 mL volumetric flasks

10 times. Then 5 mL aliquot was taken from each 25 mL leachate containing volumetric

flasks and transferred to a 50 mL centrifuge tube for microwave digestion following the same

procedure as described in the batch adsorption system. After having assessed the saturation

point of the column, the procedure was optimized by adjusting the adsorbent dose, diameter

of the column used and pH. Among the individual adsorbents and their mixture, the

combination of activated carbon and chitosan was selected based on its high Pt removal

efficiency for tested Pt species ([PtCl6]2-

and oxaliplatin).

The optimized columns were packed with 10 g of activated carbon and 2.3 g of chitosan. The

flow rate was set at 26 mL min-1

and bed volume of the adsorbent was 30 mL. 1,000 mL of

24

solution with a concentration of 100 µg Pt L-1

was percolated through the filter. The percolate

was collected at the out-let and every 50 mL of the solution was sampled in a volumetric

flask (i.e. 20 times). Control samples were taken directly from the solution without passing

the filtering media. Sample analysis was similar as in the batch adsorption test described

above. All experiments were conducted at a temperature of 25 oC to be around the average

natural temperature of human urine. The relative remaining concentration was calculated, and

media saturation and breakthrough volume were determined. The filtering media were

regenerated by using 2 M NaOH. Each filter medium was used maximum three times.

4.4.3 Desorption experiment

Platinum recovery and regeneration of the biomaterials was done by doing desorption

experiment. 500 mL of NaOH was flushed through the media containing sorbed platinum and

filtered by gravity and the percolate was collected in the out let. Then 5 mL sample was taken

from the percolate and digested with 3 mL HCl and 1 mL HNO3 acid and analysis was done

with ICP-MS by following the same procedure as described in the batch adsorption system.

Recovery rate was calculated by using this formula.

100*)adsorbedmount

desorbedamount ( (%) ratio desorption a

Pt

The overall mass balance equation was done as follows.

pMMMM dei

where, Mi is the total mass of Pt in the influent (µg), Me is the Pt in the eluate or effluent

(µg), Md is Pt temporarily sorbed to the biomaterial (µg) and Mp is Pt that is permanently

sorbed (µg) and, without complete destruction of the biomaterial, can only be derived from

other parameters. .

4.4.4 Characterization of biomaterials

Total carbon that could leach from the biomaterials was measured. To a mass of 0.1 g of each

biomaterial Milli-Q water was added to a L/S ratio of 200 and the mixture was shaken for 24

h. The obtained mixture solution was filtered (Macherey—Nagel, Germany). The control

group was prepared using Milli-Q water without adding biomaterials, but treated similarly as

25

the samples. Eventually, the filtrate was analysed for TOC content using a TOC analyser

(Shimadzu, Colombia) after calibration with low and high concentrations of total carbon (10

mg L-1

and 100 mg L-1

TC).

4.4.5 Pt analysis

All Pt concentrations in solution were determined using inductively coupled plasma- mass

spectrometry (ICP-MS) (PerkinElmer Sciex. Waltham, MA). Indium in 1% HNO3 was added

as internal standard.

4.4.5.1 Operating condition of ICP-MS

Before analysis of every batch of samples the instrument was tuned. Operational conditions

are described in Table 6.

Table 6: ICP-MS instrument conditions

Isotopes measured 195

Pt, 115

In

Nebulizer gas flow 0.68 L min-1

Ion lens voltage 7.7 V

Scan Mode peak hopping

Dwell time per AMU (ms) 75

Integration time 2250

Replicates 3

4.4.6 Quality control

All samples, except for the column test, were analysed in triplicates. All standards and CPC

were labelled, closed and stored on the recommended temperature to avoid any species

conversion during storage and handling. Analytical results generated by ICP-MS were only

accepted when the determination coefficient of the calibration curve exceeded 0.9990.

Moreover, one standard from every batch in low concentration range was used for quality

control with only 5-10% deviation tolerated.

26

5. Results

5.1 Characterization of the biomaterials

The tested biomaterials showed considerable differences in the amount of total organic

carbon they released in the solution. Coffee husk released the highest amount, i.e 3.9 g C kg-

1, while activated carbon released the lowest amount 1.05 g C kg

-1. The other biomaterials

were found to release organic matter amounts in between this range (Fig. 5).

Figure 5: TOC released by biomaterials using synthetic urine at pH 6, after 24h shaking

at 25 oC (mean + standard deviation, n=3).

5.2 Batch adsorption studies

5.2.1 Sorption to biomaterials

The pH is one of the critical parameters to be evaluated concerning adsorption. The effect of

pH on platinum removal efficiency of the biomaterials was studied by conducting the

sorption test in a wide pH range (from 5 to 11). Initially, pH 7, which is practical common pH

value for human urine, was selected to see its influence on adsorption of each Pt species.

Generally, higher sorption of Pt by activated carbon was observed (Fig. 6). Moreover, the test

statistics showed that there is a significant difference (P-value < 0.001) in the sorption of Pt

species at this pH level between activated carbon, chitosan and biochar, except for

carboplatin (P-value > 0.05) (Table 7).

27

The multiple comparison test done by post-hoc analysis (Tukey HSD) revealed that the

removal of [PtCl6]2-

and cisplatin by activated carbon, chitosan and biochar was found

statistically significant (P-value < 0.05). On the contrary, all biomaterials removed

carboplatin with out significant difference (P-value > 0.05). Eventually, oxaliplatin removal

was significant only for activated carbon (P-value > 0.05) (Annex I).

Table 7: Data generated by ANOVA test after comparing different Pt species sorption on

biomaterials

Sum of Squares Df Mean Square F Sig.

[PtCl6]2-

Between Groups 6243,920 2 3121,960 619,027 < 0.001

Within Groups 30,260 6 5,043

Total 6274,180 8

Cisplatin

Between Groups 821,376 2 410,688 79,029 < 0.001


Total 852,556 8

Carboplatin

Between Groups 197,167 2 98,583 4,355 ,068


Total 332,980 8

Oxaliplatin

Between Groups 7176,442 2 3588,221 397,660 < 0.001


Total 7230,582 8

28


, cisplatin, carboplatin and oxaliplatin by activated

carbon, chitosan and biochar at pH 7 using synthetic urine (mean + standard deviation, n=3).

The initial Pt concentration was 100 µg L-1

.

The combined use of biomaterials in a 50-50 mixture ratio at pH 7 was also studied.

Combinations of biomaterials with activated carbon resulted in the highest relative removal

of platinum. The minimum relative removal of all combinations with activated carbon was

56% while other combinations such as those of chitosan with coffee husk and saw dust, were

found to not remove more than 8% (Fig. 7).


by different

combinations of biomaterials (in a 50-50

mixture ratio) using synthetic urine at pH 7 (mean + standard deviation, n=3). The initial Pt


.

The influence of pH on the sorption of Pt species by the biomaterials was assessed. The

highest relative sorption of [PtCl6]2-

by activated carbon (78%) was achieved at pH 7. At pH 6

and 8, similar values were obtained, but the removal efficiency started to decrease

significantly when the pH exceeds 8. As also observed for activated carbon, the sorption

efficiency of chitosan was also higher at pH 6 and 7 (Fig. 8), with the relative removal of

platinum being 62% at pH 6 and 61% at pH 7. From pH 8 onwards the relative removal

started decreasing. It even decreased below 20% at high pH (10 and 11) (Fig. 8).

29


by activated carbon, chitosan and biochar at different

pH levels using synthetic urine (mean + standard deviation, n=3). The initial Pt concentration

was 100 µg L-1

The effect of pH on sorption of [PtCl6]2-

by activated carbon, chitosan and biochar was

statistically evluated. The overall test result showed that pH had a very strong influence on

sorption efficiency of biomaterials and its effect was highly significant (P-value < 0.001).

The post hoc test (tamhane) further analyse the groups that significantly differ from the

others. The sorption of [PtCl6]2-

by activated carbon did not differ significantly (P-value >

0.05) between pH 6, 7 and 8. Similar results were also found for chitosan. Biochar sorption

efficiency differed significantly only at pH 7 and 9 (Annex II). At other pH values no

significant differences were observed (Table 8). Moreover, the adsorption data also showed

that higher removal efficiencies were observed around the neutral pH (Fig. 8).

30

Table 8: Data generated by ANOVA test to tudy differences in sorption effectiveness of

[PtCl6]2-

between different pH levels for different biomaterials (activated carbon, chitosan

and biochar)

Sum of Squares Df Mean Square F Sig. Activated

carbon

Between Groups 6100,563 6 1016,761 89,693 < 0.001

Within Groups 158,704 14 11,336

Total 6259,267 20

Chitosan Between Groups 8449,046 6 1408,174 402,939 < 0.001


Total 8497,972 20

Biochar Between Groups 290,890 6 48,482 15,844 < 0.001


Total 333,730 20

The relative removal of cisplatin was slightly favoured at pH 7 and 9 for all biomaterials,

except biochar, which showed a rather low pH dependency. Relative removal of cisplatin by

activated carbon was 42.6% at pH 9, while chitosan managed to remove 34.3% at the same

pH (Fig. 9).

As Anova test was also conducted to determine the effect of pH on the sorption of cisplatin

by activated carbon, chitosan and biochar. The test statistics showed that there is a significant

difference between pH levels upon adsorption by activated carbon and chitosan (P-value <

0.05) while adsorption of Pt on biochar was not affected by pH (Table 9).

The Tamhane post hoc test showed that there was a significant difference (P-value < 0.05) in

sorption of cisplatin onto activated carbon between pH 5 and pH 9. On the other hand, for

chitosan, the only pH value that showed significant difference (P-value < 0.05) was pH 11.

At other pH values, adsorption of cisplatin did not vary significantly. The effect of pH was

not statistically significant for biochar (P-value > 0.05) (Annex III).

31


cisplatin between different pH levels for different biomaterials (activated carbon, chitosan

and biochar)

Sum of Squares df

Mean

Square F Sig.

Activated

carbon

Between Groups 482,250 3 160,750 11,620 ,003


Total 592,917 11

Chitosan Between Groups 1057,583 3 352,528 7,527 ,010


Total 1432,250 11

Biochar Between Groups 277,667 3 92,556 1,569 ,271


Total 749,667 11

Figure 9: Relative removal of Cisplatin by activated carbon, chitosan and biochar at different

pH levels using synthetic urine (mean + standard deviation, n=3). The initial Pt concentration

was 100 µg L-1

32

The sorption efficiency of the biomaterials was also tested for carboplatin. The relative

removal of carboplatin at pH 7 was 56.6%, 45.9% and 46.6% for activated carbon, chitosan

and biochar, respectively (Fig. 10). In addition, the differences in sorption between the

different pH levels were tested statistically and the result did not reveal any significant

difference in sorption of carboplatin by activated carbon or chitosan between the pH values

tested. However, sorption on biochar was slightly different at a pH level of 9 and 11 (P-value

< 0.05). In general, sorption of carboplatin is less dependent on pH compared to other Pt

species.

Table 10: Data generated by ANOVA test to study differences in sorption effectiveness

of carboplatin between different pH levels for different biomaterials (activated carbon,

chitosan and biochar)


Activated

carbon

Between Groups 137,667 3 45,889 2,673 ,118


Total 275,000 11

Chitosan Between Groups 34,917 3 11,639 ,419 ,744


Total 256,917 11



Total 550,250 11

Figure 10: Relative removal of carboplatin by activated carbon, chitosan and biochar at



.

33

Finally, the relative removal of oxaliplatin was examined under similar circumstances.

Interestingly, a very high relative removal of oxaliplatin was achieved by activated carbon.

The average relative removal of oxaliplatin by activated carbon was 95.0%. Furthermore,

chitosan and biochar removed 38% and 32% of oxaliplatin, respectively (Fig. 11).

A similar statistical test was conducted to test any effect of pH on adsorption. The result

revealed that the only significant difference (P-value < 0.05) was seen for activated carbon at

pH 5, where sorption is lower than at all other pH values tested. Adsorption on chitosan and

biochar was not found to be dependent on pH (Table 11).


oxaliplatin between different pH levels for different biomaterials (activated carbon,

chitosan and biochar)


Activated

carbon

Between Groups 755,333 3 251,778 1510,667 < 0.001

Within Groups 1,333 8 ,167

Total 756,667 11

Chitosan Between Groups 291,000 3 97,000 1,130 ,393


Total 977,667 11



Total 220,250 11

34

Figure 11: Relative removal of oxaliplatin by activated carbon, chitosan and biochar at



.

Present table 12 summerizes the relative removal of [PtCl6]2-


oxaliplatin by activated carbon, chitosan and biochar at pH 7.

Table 12: Relative removal (%) of platinum species using different biomaterials at pH 7

Platinum compounds

Biomaterials

Activated carbon Chitosan Biochar

[PtCl6]2-

78.5 + 0.7 60.4 + 0.8 15.6 + 4.9

Cisplatin 46.2 + 1.9 34.3 + 2.5 23.2 + 2.9

Carboplatin 56.6 + 3.4 45.9 + 5.7 46.6 + 4.3

Oxaliplatin 95.0 + 0.8 38.5 + 2.7 32.7 + 4.4

Total average 69.1+ 21.9 44.8 + 11.5 29.6 + 13.4

35

5.2.2 Sorption to PMO-SH

The platinum sorption efficiency of the thiol containing Periodic Mesoporous Organosilica

(PMO-SH and PMO-SH(50%)) was also studied in a batch adsorption test. The result of the

study indicates that PMO-SH managed to remove 82.4%, 74.3%, 35% and 88.4% for

[PtCl6]2, cisplatin, carboplatin and oxaliplatin, respectively at pH 6. On the other hand the

removal efficiency of PMO-SH(50%) was 61.8%, 79.3%, 21% and 65.6% for [PtCl6]2-

,

cisplatin, carboplatin and oxaliplatin, respectively (Fig. 12).

Moreover, Mann-Whitney U and unpaired t-test were conducted to determine any difference

between the sorbents in their sorption efficiency. The test result revealed that there is a

significant difference between PMO-SH and PMO-SH(50%) in sorption of oxaliplatin (P-value

< 0.05) only. Other Pt species did not show any significant difference (Table 13 and 14).

Table 13: Data generated by Mann-Whitney U test to study differences in sorption effectiveness

of [PtCl6]2-

by PMO-SH and PMO-SH(50%)

Platinum

species

Statistical

test

Group name Median 25% 75% P-value Significance

[PtCl6]2-

Mann-

Whitney U

PMO-SH 82.918 78.764 85.583 0.800 Insignificant

PMO-SH(50%) 61.687 40.086 83.287

Normality Test was done using (Shapiro-Wilk)

Table 14: Data generated by unpaired t-test to study differences in sorption effectiveness of

cisplatin, carboplatin and oxaliplatin by PMO-SH and PMO-SH(50%)

Platinum

species

Statistical

test

Group name Mean Std Dev SEM P- value Significance

Cisplatin

Unpaired

t-test

PMO-SH 74.554 19.557 11.291 0.720

Insignificant

PMO-SH(50%) 80.351 3.530 2.496

Carboplatin

“

PMO-SH 35.045 10.787 6.228 0.311 Insignificant

PMO-SH(50%) 20.978 15.799 11.171

Oxaliplatin

“

PMO-SH 88.437 8.279 4.780 0.039 Significant

PMO-SH(50%) 65.587 3.854 2.725

Normality Test was done using (Shapiro-Wilk)

36

Fig. 12: Relative removal of [PtCl6]2-

and CPC by PMO-SH and PMO-SH(50%) in Milli-Q

water at pH 6 and L/S=1,000, after 24 h mixing and centrifugation at 2,500 rpm for 10 min

(mean + standard deviation, n=3). The initial Pt concentration was 100 µg L-1

.

5.2.3 Effect of concentration on sorption

5.2.3.1 Sorption isotherm

The effect of the initial concentration on the sorption of platinum to the biomaterials was

studied by using different Pt concentrations in batch experiments. The isotherm expresses the

distribution of target compounds ions between solution and the biomass after reaching

equilibrium.

The Freundlich isotherm model was constructed for the selected biomaterials and the values

of Kf, n and R2 are presented in Table 15. In all cases the Freundlich model was found to fit

with a very high correlation coefficient.

37

Table 15: Sorption isotherm parameters

Biomaterial Pt species Freundlich isotherm model

Kf (µg1-1/n

g-1

) n R2

Activated

carbon

[PtCl6]2-

0.14+ 0.004 1.09+ 0.04 0.999

Cisplatin 0.14 + 0.05 1.07+0.06 0.998

Carboplatin 0.11+ 0.01 0.97+ 0.02 0.999

Oxaliplatin 0.19+0.002 1.00+0.001 1.000

Chitosan

[PtCl6]2-

0.06+0.014 1.07+0.03 0.998

Cisplatin 0.08+ 0.017 1.04+0.04 0.999

Carboplatin 0.14+ 0.08 1.34+0.06 0.999

Oxaliplatin 0.73+0.02 1.02+0.05 0.998

Biochar

[PtCl6]2-

0.06+ 0.014 1.06+0.03 0.999

Cisplatin 0.05+0.02 0.98+0.05 0.999

Carboplatin 0.06+0.006 0.90+0.02 0.999

Oxaliplatin 0.16+ 0.13 1.48+ 0.14 0.971

The sorption isotherm test for [PtCl6]2-

showed that there was a difference in the amount of Pt

adsorbed among the different biomaterials. For example, at an initial concentration of 500 µg

L-1

the amount of [PtCl6]2-

adsorbed by activated carbon, chitosan and biochar was found to

be 31 µg g-1

, 16 µg g-1

and 11 µg g-1

respectively.

As presented in Figure 14, the adsorption isotherm of the amount of Pt adsorbed by

biomaterial (qe) versus equilibrium concentration (Ceq) had very good fit for all biomaterials

(Fig.13).

38

Activated carbon

Freundlich fit

Ceq (µg Pt L-1)

0 200 400 600 800

qe

(µg P

t g-1

bio

ma

teri

al)

0

20

40

60

80

100

Chitosan

Freundlich fit

Ceq (µg Pt L-1)

0 200 400 600 800

qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

50

60

Biochar

Freundlich fit

Ceq (µg Pt L-1)

0 200 400 600 800 1000

qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

Figure 13: Adsorption isotherms for adsorption of [PtCl6]

2- in synthetic urine on activated

carbon (upper left), chitosan (upper right) and biochar (bottom) (mean + standard deviation,

n=3).

Unlike that of [PtCl6]2-

, the adsorption isotherm study for CPC only considered initial

concentrations of 1 µg L-1,

20 µg L-1

, 100 µg L-1

, 200 µg L-1

and 500 µg L-1

as the amount of

available CPC products was limited. However, the concentration range was still large to

cover the expected concentrations of platinum in hospital effluent or cancer patient’s urine on

the one hand and to construct a complete isotherm on the other hand. For the initial cisplatin


, activated carbon, chitosan and biochar adsorbed 45 µg g

-1, 31 µg

g-1

and 28 µg g-1

, respectively (Fig. 14).

39

Activated carbon

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

50

60

Chitosan

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

Biochar

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

Figure 14: Adsorption isotherms for adsorption of cisplatin in synthetic urine on activated

carbon (upper left), chitosan (upper right) and biochar (bottom) (mean + standard deviation,

n=3).

Adsorption isotherms were also calculated for carboplatin with similar initial concentrations

as for cisplatin, except that one intermediate concentration of 52.5 µg L-1

was added (Fig.

15). For an initial concentration of 500 µg L-1

, activated carbon, chitosan and biochar

adsorbed 63 µg g-1

, 44 µg g

-1 and 57 µg g

-1 respectively, which was higher than the capacity

seen for cisplatin.

40

Activated carbon

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500 600

qe

(µg P

t g-1

bio

ma

teri

al)

0

20

40

60

80

100

Chitosan

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500 600qe

(µg P

t g-1

bio

ma

teri

al)

0

10

20

30

40

50

60

70

Biochar

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500 600

qe

(µg P

t g-1

bio

ma

teri

al)

0

20

40

60

80

Figure15: Adsorption isotherms for the adsorption of carboplatin in synthetic urine on

activated carbon (upper left), chitosan (upper right), biochar (bottom left) (mean + standard

deviation, n=3).

Eventually, the adsorption isotherm test was conducted also using oxaliplatin. The initial

concentrations of oxaliplatin tested were identical to those used for cisplatin. The amounts of

platinum adsorbed by activated carbon, chitosan and biochar were 89 µg g-1

, 30 µg g-1

and 22

µg g-1

, respectively, for the initial concentration of 500 µg L-1

(Fig. 16). All sorption

isotherms clearly illustrate that for all platinum species tested activated carbon had the

highest potential for sorption.

41

Activated carbon

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teia

ls)

0

20

40

60

80

100

Chitosan

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teia

ls)

0

10

20

30

40

Biochar

Freundlich fit

Ceq (µg Pt L-1)

0 100 200 300 400 500

qe

(µg P

t g-1

bio

ma

teia

ls)

0

5

10

15

20

25

30

35

Figure 16: Adsorption isotherms for the adsorption of oxaliplatin in synthetic urine on

activated carbon (upper left), chitosan (upper right), biochar (bottom) (mean + standard

deviation, n=3).

5.2.4 Fixed bed adsorption study (continuous flow test)

5.2.4.1 Screening column experiment

The column experiment was performed using [PtCl6]2-

in a small sized column (⌀ 2.40 cm) to

determine the combination of biomaterials that could achieve highest relative removal and

evaluate parameters including adsorbent dose, pH, flow rate, and diameter of the column that

should be fulfilled for optimization.

42

The experiment was done by using a column packed with activated carbon, chitosan and

biochar, both separately and combined. All tests were done using synthetic urine. Moreover,

one extra column was set up with Milli-Q water to evaluate the effect of matrix on sorption.

The column packed with activated carbon was able to remove [PtCl6]2-

by 82.7% for the first

25 mL elution volume while chitosan and biochar removed 65.5% and 16.7%, respectively.

The removal of platinum by the column packed with activated carbon then dropped to reach

close to its saturation after percolating 75 mL of [PtCl6]2-

solution, while chitosan and biochar

were already saturated after only 25 mL.

Among the combinations of biomaterials, activated carbon and chitosan managed to achieve

very low relative remaining concentration (high removal efficiency) and were therefore

selected to further testing. Results of the combination of activated carbon and chitosan

showed that removal efficiency of [PtCl6]2-

was 87.5% during the first elution volume of 25

mL. The ANOVA statistical test result showed that there is a significant difference (P-value <

0.05) among the three combinations of biomaterials tested. Tukey HSD test further confirmed

that activated carbon and chitosan combnation is significantly different (P-value < 0.05) from

the other two combinations. Moreover, Figure 17 showed that this combination showed

overall a better relative removal for all [PtCl6]2-

solutions treated.



through a column containing different combination of

biomaterials as function of eluate volume (preconditioned with 0.5 M HCl and Milli-Q water,

bed volume = 10 mL, initial Pt concentrations 100 µg L-1

).

43

Furthermore, the rationale of using a dual bed adsorption column instead of single bed

column was also evaluated using CPC. Cisplatin and oxaipaltin were used to examine

whether the dual bed also favors sorption of CPC besides [PtCl6]2. The relative removal of

oxaliplatin by the bed containing activated carbon and chitosan was 98% for the first 50 mL

elution volume, while the column packed with activated carbon alone removed 85.8% for the

same elution volume. The removal efficiency of the dual bed consisting of activated carbon

and chitosan was higher than the single bed for the entire volume of oxaliplatin solution

percolated (Fig. 18). Statistical tests showed that this difference in removal efficiency was

also significant (P-value < 0.001) (Table 16).

Table 16: Data generated by Mann-Whitney U Test to compare the single and dual bed

column use for sorption of oxaliplatin in a continous flow system

Group n Missing Median 25% 75% Sig.

Dual bed column 7 0 96.065 91.974 97.365 < 0.001

Single bed column 7 0 69.554 68.437 70.549

Mann-Whitney U Statistic = 0.000

T = 77.000, n = 7

Figure 18: Relative remaining concentrations of oxaliplatin

in synthetic urine by percolating

through a filter contained of chitosan and activated carbon (blue) and activated carbon (red)

as function of eluate volume (column preconditioned with 0.5 M HCl and Milli-Q water),

flow rate and bed volume for dual bed column was 26 mL min-1

and 30 mL, respectively

while for single bed column 68 mL min-1

and 42 mL, respectively. Initial Pt concentration

was 100 µg L-1

.

44

The same dual bed was also examined for sorption of cisplatin during percolation of 350 mL.

An unpaired t-test showed that there was a signdificant difference in sorption between them

but not very strong since the test statistics generated a P-value exactly on the cut off point (P-

value = 0.05) (Table 17).

Table 17: Data generated by unpaired t-test after comparing the single and dual bed column

use for sorption of cisplatin in a continuous flow system

Group name N Missing Mean Std Dev SEM sig.

Single bed 7 0 25.997 17.460 6.599 0.05

Dual bed 7 0 45.936 25.201 9.525

t = -1.721, Df = 12

5.2.5 Column optimization

5.2.5.1 Dose determination

The maximum sorption capacity of the biomaterials was determined in a batch adsorption

study prior to conducting to a column test. This was done by mixing the biomaterial in

different ratio with 20 mL of [PtCl6]2-

solution containing 100 μg Pt L-1

. The highest platinum

removal efficiency was achieved at 0.5 g in a L/S ratio of 40 (Fig. 19).

45

Figure 19: Relative remaining concentration of Pt in batch adsorption study with synthetic

urine containing [PtCl6]2-

and using activated carbon and chitosan (50-50) as adsorbent at pH

7, after 24h shaking, with initial Pt concentrations of 100 µg L-1

) (mean + standard deviation,

n=3).

5.2.5.2 Optimum pH determination

As one of the parameters that influence sorption, the optimum pH for the continuous flow test

was determined. From batch experiments, the best removal was generally found in the pH 7

to 9 region, while not being significantly different with in this pH region. Continuous flow

tests therefore were executed at pH values of 7, 8 and 9. Sorption of Pt by the packed column

was found to be slightly higher at pH 7 and the difference became more clear as the eluate

volume increased. The relative removal was found to be 99.2%, 98.3% and 97.3% for pH 7, 8

and 9, respectively, after the first 25 mL elution volume (Fig. 20).



through a column containing activated carbon and

chitosan as function of eluate volume (column preconditioned with 0.5 M HCl and Milli-Q

water, flow rate = 26 mL min-1

, bed volume = 30 mL and initial Pt concentration of 100 µg L-

1).

46

5.2.5.3 Matrix effect

The sorption process can also be affected by the composition and characteristics of the

aqueous medium due to the non-selective nature of adsorption. In this study, two different

media, synthetic urine versus Milli-Q water, were compared in a column test under similar

test conditions.

The relative removal of platinum was found to be 93.2% in Milli-Q water and 92.7% in

synthetic urine for the first 50 mL (Fig 21). With increasing elution volume, a more

significant difference in removal efficiency was observed. Platinum removal dropped to 70%

for the synthetic urine solution, while it remained 92.4% in Milli-Q water. After 250 mL, still

more than 83% was removed from the solution in Milli-Q water, whereas the removal from

synthetic urine decreased already to 50% after passing 100 mL solution.

According to the Mann-Whitney U Test, there was a very significant difference in sorption

(p-value < 0.001), during the treatment of [PtCl6]2-

between synthetic urine and Milli-Q water

(Table 18).

Table 18: Data generated by Mann-Whitney U Test to compare sorption of [PtCl6]2-

contained

in synthetic urine and Milli-Q water by a column packed with activated carbon and chitosan.

Group N Missing Median 25% 75% Sig.

Milli-Q water 10 0 95.283 89.992 96.405

< 0.001 Synthetic Urine 10 0 48.289 35.098 63.425


t = 152.000, n = 10

47


in both synthetic urine (circular dot

line) and Milli-Q water (rectangular dot line) following percolation through a filter contained

of chitosan and activated carbon (50:50) as function of eluate volume (column preconditioned

with 0.5 M HCl and Milli-Q water), flow rate = 26 mL min-1

, bed volume = 30 mL and initial

Pt concentrations of 100 µg L-1

.

The role of optimization of the filter media was also assessed by making comparison with the

removal efficiency of a non-optimized column (Fig. 22). The fliter media was optimized by

determining the optimum dose of biomaterials, adjusting pH, and flow conditions by using a

different size column. The flow rate and bed volume for non-optimized were 7 mL min-1

and

10 mL, respectively while optimized colum adjusted at a flow rate of 26 mL min-1

and bed

volume of 30 mL. Moreover, the diameters of the columns were ⌀ 2.4 cm and ⌀ 3.10 cm for

non-optimized and optimized column, respectively.

The removal efficiency for the first 25 mL elution volume was 92.7% for the optimized and

87.5% for the non-optimized column. In addition, the non-optimized column reached to its

saturation after treating only 75 mL of [PtCl6]2-

while, the optimized column treated 225 mL

before reaching saturation. Moreover, the change in sorption as a result of optimization of the

column was checked statistically by using Mann-Whitney U Test and a significant difference

was observed (p-value < 0.01) (Table 19).

48

Table 19: Statistical data generated by Mann-Whitney U Test to compare [PtCl6]2-

sorption

from synthetic urine between an optimized and a non-optimized column packed with

activated carbon and chitosan

Group N Missing Median 25% 75% Sig.

After optimization 10 0 90.885 85.839 91.955

0.002 Before optimization 10 0 48.289 35.098 63.425


t = 146.000, n = 10


in synthetic urine following

percolation through a non-optimized (circular dot line) and optimized (rectangular dot line)

filter contained of chitosan and activated carbon (50:50), as function of eluate volume

(column preconditioned with 0.5 M HCl and Milli-Q water) flow rate and bed volume for non

optimized was 7 mL min-1

and 10 mL, respectively, while 26 mL min-1

and 30 mL for

optimized column, respectively. Initial Pt concentration was 100 µg L-1

.

5.2.6 Full scale column experiment

a) [PtCl6]2-

The selected biomaterials (combination of activated carbon and chitosan) were further tested

with larger diameter column size (⌀ 3.10 cm) to accommodate higher adsorbent dose. The

experiment was conducted at pH 7, which was previously found to be the optimum pH. As in

49

the screening test, the initial concentration for Pt in the influent was 100 µg L-1

. The

combination of activated carbon and chitosan packed in the column managed to remove

[PtCl6]2-

by 96.8% during the first 50 mL elution volume. The relative removal of the dual

bed after treating 50% (500 mL) of [PtCl6]2-

solution was found 24.4% (Fig. 23). Bed

exhaustion (> 0.9) was reached after treating all [PtCl6]2-

solution (1,000 mL) (Fig. 24).



& CPC through a column of activated carbon and chitosan

as function of eluate volume (column preconditioned with 0.5 M HCl and Milli-Q water, flow

rate = 26 mL min-1

, bed volume = 30 mL and initial Pt concentration of 100 µg L-1

).

b) Cisplatin

The same combination of biomaterials (activated carbon and chitosan) was also tested for

removal of cisplatin. The relative remaining concentration of platinum after the first 50 mL

elution volume was 14.3%, which is higher than when using [PtCl6]2-

for the same elution

volume. Moreover, the relative removal after treating 50% (500 mL) of the effluent dropped

to 6.4% (Fig. 23). Its relative removal became nearly constant and stable after treating 600

mL. The difference in relative removal after reaching the saturation point was below 0.26%

(removal between 600 and 900 mL). The calculated Ce/Co ratio in the saturation region was

found to be 0.85 and constant, which proved that the media became saturated (Fig 24).

50

c) Carboplatin

The combined biomaterial consisting of activated carbon and chitosan was tested for

carboplatin removal and the result revealed that the relative remaining concentration after

collecting the first 50 mL elution volume was 4.7%, coinciding with a removal of 95.3%

(Fig. 23). The relative removal decreased slightly as the elution volume increased and did not

show a clear saturation point. The relative removal efficiency after treating 1,000 mL was

found 12.2%. Filter media exhaustion was reached after treating the entire effluent percolated

for [PtCl6]2-

, cisplatin and carboplatin. Media exhaustion is considered when Ce/Co value is >

90% (Fig. 24).

Figure 24: Ratio of relative remaining concentration (Ce) to the concentration of the control

(Co) during percolation of synthetic urine containing [PtCl6]2-


oxaliplatin through a column of activated carbon and chitosan as function of eluate volume

(column preconditioned with 0.5 M HCl and Milli-Q water, flow rate = 26 mL min-1

, bed

volume = 30 mL and initial Pt concentration of 100 µg L-1

).

d) Oxaliplatin

Eventually, the removal of oxaliplatin by sorption using the same combination of

biomaterials (activated carbon and chitosan) was tested. The relative remaining concentration

of the first 50 mL elution volume was 1.9%. Unlike the above three platinum solutions tested,

oxaliplatin could be very effectively removed from the solution. After percolating 1,000 mL

of the solution, still 77.3% of the oxaliplatin removal was found and no saturation occurred at

this elution volume (Fig. 23 and 24).

51

In general, sorption of Pt compounds by the filter meida was higher for oxaliplatin and

followed in decreasing order for carboplatin > [PtCl6]2-

> cisplatin (Fig. 23). Moreover, the

removal efficiency of the optimized column for the different Pt species was also tested

statistically. The ANOVA test result indicated that there is a significant difference (P-value <

0.001) in the removal of [PtCl6]2-

, cisplatin, carboplatin and oxaliplatin (Table 20).

Table 20: Statistical data generated by ANOVA test to compare sorption of [PtCl6]2-

,

cisplatin, carboplatin and oxaliplatin from synthetic urine on

the optimized column packed

with activated carbon and chitosan

Group Sum of Squares Df Mean Square F Sig.

Between Groups 49527,138 3 89.992 41,026

< 0.001 Within Groups 30582,750 76 35.098

Total 80109,888 79

In addition, the differences among the groups were further tested by post-hoc statistical

analysis and showed that there was a significant difference (P-value < 0.001) in the sorption

of oxaliplatin versus the other three Pt species. Moreover, sorption of carboplatin and

cisplatin differ significantly (P-value < 0.05). Any other combination was found insignificant

(P-value > 0.05) (Annex- IV).

5.2.7 Desorption study

A desorption study was conducted to assess the percent recovery of Pt after sorption by the

biomaterial followed by regeneration of the media for reuse. The result showed that 91.7% of

the temporarily adsorbed Pt was successfully recovered, so 8.3% of Pt can be considered as

being permanently adsorbed to the biomaterial.

52

6. Discussion

6.1 Introduction

The application of biomaterials for heavy metal removal and recovery becomes economically

and environmentally attractive. Therefore, our study aimed to assess the potential of selected

biomaterials for platinum removal from human urine, which could be applied and further

upgraded for recovery of this precious metal widely used in hospitals to treat cancer.

6.2 Screening of biomaterials

Finding the right sorbent with high sorption efficiency is necessary. One of the factors

associated with sorption efficiency of biomaterials is the presence of high soluble organic

carbon that leaches into the solution. High organic carbon content is related to high metal

sorption capacity (Miretzky et al., 2005) but leaching of soluble organic carbon could release

the adsorbed metals into the solution during filtration. Among the biomaterials tested, coffee

husk released the highest amount of TOC (3.9 g C kg-1

) and comparatively its [PtCl6]

2-

removal efficiency was found to be the lowest, i.e 21.1%. On the other hand, activated

carbon, releasing the lowest organic carbon amounts into the solution also, managed to

remove all Pt species at a higher efficiency. The biomaterials releasing too high TOC

amounts can not be good candidates to act as adsorbent in continuous flow systems. This, in

combination with removal efficiency, was taken under consideration when biomaterial was

selected for the column experiments. Activated carbon and chitosan were among materials

releasing the the lowest TOC amounts to the solution.

6.3 Potential use of materials for Pt sorption

6.3.1 Biomaterials

In general, the [PtCl6]

2- removal efficiency differed significantly between the biomaterials

tested in our study (P-value < 0.001), except between coffee husk and saw dust and biochar at

pH 7. Differences in sorption efficiency of biomaterials are mainly linked to their nature,

specific surface area, average pore diameter, selectivity, available binding sites etc. (Buekens

and Zyaykina, 2012). Activated carbon showed the highest removal for all Pt species with a

53

maximum sorption achieved for oxaliplatin (95.0%). Many studies noted that activated

carbon is by far the most preferred in precious metal recovery due to very high capacity and

adsorption rate. According to Sharififard et al. (2012) activated carbon was able to remove

98% of Pt at pH 2. The same study also showed that, although activated carbon has high

sorption capacity, coating with bio-polymer (chitosan based) managed to increase its capacity

towards 45.5-52.6 mg g-1. In our study, it was also found out that activated carbon adsorbed

the highest amount of [PtCl6]2-

and CPC at pH 7. The amount of Pt adsorbed by activated

carbon was found to avry in a range of 30-89 µg g-1

for Pt solution with an initial


.

Activated carbon is mainly considered as an ideal adsorbent for recovery of precious metal

due to its high internal surface area (Lenntech, 1998). Besides its use for metal recovery, it is

also very effective in adsorption of dyes. However, its use is often compromised by problems

related to regeneration and costs (Kismir and Aroguz, 2011). Hence, its use in combination

with other biomaterials may become interesting. Our study also showed that biomaterials

mixed with activated carbon in a 50-50 ratio showed a significant change in [PtCl6]2-

removal. It seems possible to maximise sorption of less costly biomaterials by mixing it with

activated carbon. For example, coffee husk, having the lowest Pt removal on its own,

managed to remove platinum by 56% when mixed with activated carbon.

Different studies are currently also being conducted with tannin and lignin based biomaterials

(coffee husk and saw dust) as potential biosorbent for sorption of different metals from

aqueous solutions. A study conducted by Naiya et al. (2009) revealed that saw dust removed

Zn(II) and Cd(II), achieving a marked reduction of 85.8% and 94.3% respectively. Moreover,

coffee husk is currently being studied by many researchers for its potential as low cost

biosorbent to remove metals from aqueous solution. Based on the study conducted by

Oliveira et al. (2008), coffee husk had a Cu(II) sorption efficiency ranging from 89 to 96%. It

also showed a good removal of Cr(VI), Cd(II) and Zn(II) in decreasing order in a pH range

between 4-7. Metal removal is mainly due to the presence of surface functional groups that

include phenolic, carboxylic, basic and lactonic groups. Generally, the lower removal of

[PtCl6]2-

and CPC in our study might be attributed to the matrix effect of the synthetic urine

and the fact that the test was done at a very low Pt concentration at which competing ions

may have a significant influence on the sorption. Moreover, the study done by Oliveira et al.

54

(2008) also indicated that coffee husk showed a slightly lower removal of metals at pH (7),

which was also used by our study.

Chitosan followed a similar pattern as activated carbon in the removal of all Pt species except

oxaliplatin. Its removal efficiency was found to decrease in the order [PtCl6]2-

> carboplatin >

oxaliplatin > cisplatin. Many studies reported that chitosan’s inorganic platinum removal

efficiency and capacity was found higher in its modified form, due to its better strength to

cope up with extremely low pH levels and protonation of amine groups that consequently

maximize Pt removal. A study conducted by Ramesh et al. (2007) showed that glycine

modified crosslinked chitosan resin had a capacity of 122 mg g-1

for Pt(IV) for an initial

concentration of 50-500 mg L-1

at pH 2, while our study found that the amount of [PtCl6]2-

adsorbed at an initial concentration of 10 mg L-1

was 0.99 mg g-1.

Results of the different

studies can not be compared since differences in the operational parameters like initial

concentration, pH and the form of chitosan, often occurs. These parameters play a key role in

sorption and determine the sorption capacity. However, data of the different studies have

proven that chitosan has a good potential for Pt removal.

The common mechanism of removal used by chitosan includes chelation, electrostatic

attraction and ion exchange (Ramesh et al., 2007). Chitosan based sorbents have a high

nitrogen content and good porosity, which promote a high sorption capacity. Nevertheless,

the non-selective nature of adsorption limits also its capacity and many studies focused on

chemical modification to solve this problem. Selective removal of metals by chitosan is based

on the type of complexing agent that comes into contact with polymeric chain structure. For

instance, sulphur and nitrogen containing ligands are the most efficient for removal of

precious metals like Pt(IV). Complexes formed between Pt(IV) and sulphur is stable (Zhou et

al., 2009).

Biochar was previously proven to have a good capacity to adsorb anions, cations and non-

polar organic compounds. Porosity, capacity and other parameters depend on the type of

biomass used and pyrolysis temperature (Ghezzehei et al., 2014). For all species, sorption of

Pt to biochar was relatively lower compared to sorption to activated carbon and chitosan,

except for carboplatin (59%), for which biochar is slightly more effective than chitosan.

There is no study so far which studied showing the use of biochar for Pt removal, but biochar

has been used in the removal of different kinds of metals from aqueous streams. A study

55

conducted by Kołodynska et al. (2012) revealed that the sorption capacity of biochar was

found higher for Pb(II). It exhibited a moderate removal for Cd(II), Cu(II) and Zn(II) (in

decreasing order) with a maximum sorption at pH 5-6. Moreover, removal of phosphate from

dairy waste water by biochar (230 µg g-1

of biochar) is considered as promising (Ghezzehei et

al., 2014). The non-selective nature of adsorption to biochar might seriously decrease the

removal efficiency during Pt removal from human urine, which also contains phosphorous

(0.7-1 kg y-1

person-1

).

Generally, all biomaterials showed better Pt removal in the neutral pH range than basic or

acidic conditions. This supports the use of biomaterials for Pt removal from human urine, as

the optimum pH range of human urine is reported in the range 6.8-7.2 (Young, 2006). Studies

showed that chitosan starts to dissolve at pH less than 5, limiting its use unless chemical

modification is done. Cross linkers like epichlorohydrin or glutaraldehyde are usually applied

to solve this problem (Fujiwara et al., 2007; Zhou et al., 2009). Another study done by Chang

et al. (2012) reported on the use of chitosan to remove 4-chlorophenol and

tetrachloroethylene from waste water at different pH levels. The optimum pH range for these

two chlorinated organics was 6 and 4, respectively. An optimal removal at neutral pH makes

application without any chemical modification possible.

In our study, the removal of [PtCl6]2-

and CPC were found to decrease in the order activated

carbon > chitosan > biochar at pH 7. The challenge to compare results of our study with those

of most other adsorption studies is that almost all of them were conducted in extremely low

pH ranges i.e 1.6-2, which occur only in special types of waste streams (see Table 4). This

pH range does not occur in most environmental samples including human urine or hospital

effluents, which is the target of our study. In our study the effect of pH on Pt sorption was not

consistent for all biomaterials in the pH range tested (5-11). The influence of pH was very

minimal during the adsorption of carboplatin (Table 9). However, adsorption of [PtCl6]2-

was

very low at higher pH (10 and 11) and higher removal was achieved at pH 6, 7 and 8. In case

of the high pH levels, the OH- ions

in the solution

compete with the target ions for adsorption

and consequently reduce the overall sorption efficiency. Conversely, at lower pH the surfaces

of biomaterials may become positively charged, facilitating the association with anionic

complexes by electrostatic attraction (Boujday et al., 2013).

56

This result is consistent with results reported by Yang et al. (2014) on the use of bamboo

biochar for sorption of metal-complex dyes. They reported adsorption to decline significantly

at higher pH. Similarly, the sorption of Pt(IV) by thiourea-modified chitosan was found

maximal at lower pH (2) due to the protonation of amine groups that induce electrostatic

attraction towards the anionic Pt complex. However, it was assumed that the HCl used for pH

adjustment increases the chloride concentration in the solution and favours more chloro-

anionic species to be attracted (Zhou et al., 2009). But, this argument doesn’t seem applicable

in our study since the amount of chloride added during pH adjustment by HCl was much

lower than the chloride already present in the synthetic urine. Moreover, our study focused

from slightly acidic to highly basic (5-11) pH range.

Another study conducted by AL-Degas et al. (2008) revealed that the highest removal of

activated carbon was measured in a pH range of 6-8. This is because the isoelectric point of

activated carbon (pHpzc) is 9 which becomes positively charged in the above mentioned pH

range, increasing the adsorption of Pt anions. The results of our study also showed that

activated carbon removed better in the same neutral pH range (6-8) as it was reported in the

results of the study done by AL-Degas et al. (2008). We can conlculde that the highest

removal efficiencies achieved by activated carbon, chitosan and biochar occur in the pH

range close to the actual pH of human urine.

6.3.2 PMO-SH

Thiol functional groups are known for their good affinity towards Pt. Studies are currently

undertaken on their potential use for Pt recovery. In our study thiol containing Periodic

Mesoporous Organosilica (PMO-SH and PMO-SH(50%) were found efficient considering the

high L/S (1000) ratio. Comparison of the Pt removal efficiency between PMO-SH and PMO-

SH(50%), of which the latter contains only half of the thiol functional groups in their structure,

showed that there is a significant difference (P-value < 0.05) in the removal of oxaliplatin

(Table 14). The removal efficiency of PMO-SH (88.4%) was found higher than PMO-SH(50%)

(65.6%) for oxaliplatin. For all other species, the difference was not statistically significant.

The general difference in removal should probably be attributed the higher sulphur content

(13.2%) in PMO-SH. The selective adsorption of thiol functionalized mesoporous silicas

(TFMS) for Pt(IV) and Au(III) was studied by Zheng et al. (2012). They reported that The

57

result of the study found out that TFMS exhibited high capacity (707 mg g-1

) for Pt(IV) and

showed strong affinity towards Pt and Au compared to other metals like Ni2+

or Cu2+

, making

them a very attractive option to recover high quality precious metals. Another study was

conducted on improving adsorption capacity and selectivity by modifying chitosan

microspheres with thiourea for interaction with Pt(IV). The removal of Pt(IV) after the first

30 min of shaking was 73% and the capacity was found 135 mg g-1

. Sulphur groups used for

modification increased both the capacity and removal efficiency of non-modified substrate

(Zhou et al., 2009).

6.4 Sorption isotherm

Sorption isotherms describe the equilibrium relationship between sorbate and sorbent that

helps to determine the capacity of sorbents for the target compound (Ho, 2006). In this study,

the effect of initial concentration on sorption and capacity of biomaterials was assessed using

Freundlih and Langmuir isotherm models. The monolayer sorption capacity (qmax) of

biomaterials, which is derived from the Langmuir model, was not determined due to the non-

satisfied behaviour and a very low correlation coefficient, indicating that the sorption data

didn’t fit the Langmuir assumption. This might be due to the very low concentrations tested

by considering only the Pt content expected in real hospital effluent and urine of cancer

patients. It is documented that low concentration adsorption data are better explained by

Freundlich than the Langmuir model (Sime "n.d.").

Among all Pt species tested, activated carbon showed the highest affinity for oxaliplatin and

carboplatin with adsorption capacities of 89 µg g-1

and 63 µg g-1

, respectively, after

equilibrium when the initial concentration was 1-500 µg L-1

. Sharififard et al. (2012) revealed

that the capacity of activated carbon determined by the Langmuir isotherm model for an

initial concentration of 50-300 mg L-1

[PtCl6]2-

was 45.5 mg g-1

. Another study conducted by

Zhou et al. (2009) showed that when using thiourea-modified chitosan microspheres for the

removal of Pt(IV), the maximum adsorption capacity calculated was 129.9 mg g-1

for an

initial concentration 10-400 mg L-1

. On the other hand in our study chitosan adsorbed

[PtCl6]2-

in 31 µg g-1

when the intial concentration of the solution was 1-500 µg L-1

. An

extended data comparison is difficult since different concentrations, pH, and L/S ratio were

used, and because the Langmuir model was not used in our study. From the sorption isotherm

58

graph, no sign of saturation was observed, except for biochar during sorption of [PtCl6]2-

and

oxaliplatin.

Data for [PtCl6]2-

and CPC were found to fit the Freundlich model well with correlation

coefficients (R2) for activated carbon, chitosan and biochar being above 0.97 (Table 15). The

preference of the data for fitting the Freundlich isotherm is probably an indication that

adsorption occurs at multiple sites or in a heterogeneous way instead of in monolayers.

Moreover, the adsorption intensity, which is represented by “n” in the Freundlich model, was

greater than 1 for most of the biomaterials, showing that adsorption matches the Freundlich

sorption principle.

6.5 Sorption test on continuous flow system

Continuous flow experiments were conducted based on the data input from batch adsorption

tests on capacity and dose. Moreover, a selection was made of best suited biomaterials and

combinations of biomaterials for the system in terms of Pt removal efficiency, saturation and

desorption.

From the screening column experiment comprising individual and combined biomaterials

used for sorption, the dual bed column made from activated carbon and chitosan was

selected. Besides comparing the Pt removal efficiency of different biomaterials, a statistical

test was also conducted to evaluate whether differences were significant or not. It confirmed

that the removal efficiency of a dual bed column composed of activated carbon and chitosan

showed a statistically significant higher value for oxaliplatin (P-value < 0.05) compared to a

single bed column containing activated carbon (Fig. 18). A possible reason why the dual bed

is more efficient is the fact that chitosan regulates the flow rate and increases the contact time

between the biomaterials and Pt species. The effect of flow rate on adsorption of Pb2+

in a

fixed bed adsorption system was studied before by Nwabanne and Igbokwe (2012). They

found that sorption decreases as the flow rate increases due to less contact time between

target compound and surface of the sorbent. Overall, this leads to the reaching of saturation

within a shorter time. Another study conducted by Chowdhury et al. (2012) using two

different flow rates and granular activated carbon showed that the sorption efficiency at the

high flow rate was lower and saturation was reached much faster than at the low flow rate.

59

Nomanbhay and Palanisamy (2005) also conducted a batch adsorption test to study removal

of chromium metal ions by chitosan coated onto acid treated oil palm shell charcoal and

compared its capacity with non-chitosan coated material. They confirmed that the chitosan

coated charcoal had a higher capacity (60.3 mg g-1

) over the non-coated one (44.7 mg g-1

).

Therefore, the use of chitosan as coating or in combination increases removal efficiency and

capacity of charcoal for adsorption of Cr(VI). The result of our study also confirmed that the

selected dual bed column showed better removal efficiency than other combinations of

biomaterials over the entire elution volume (250 mL) (Fig 17).

The selected combination of biomaterials for the fixed bed adsorption required optimization

to improve sorption efficiency and to control the media from reaching saturation too quickly.

Therefore, we checked whether there was a difference in sorption efficiency following

optimization of the column. The result of this evaluation showed that there is a statistically

significant difference in the removal of [PtCl6]2-

(P-value < 0.001) between the optimized and

non-optimized column (Table 19).

The removal efficiency of the optimized column for oxaliplatin, carboplatin, [PtCl6]2-

and

cisplatin were 98.0%, 95.4, 96.8% and 85.8%, respectively for the first 50 mL elution

volume. In general, removal efficiency was highest for oxaliplatin, followed by, in decreasing

order, carboplatin, [PtCl6]2-

and cisplatin.

Regarding the high removal efficiency of oxaliplatin, a study was conducted by Hann et al.

(2005) on the stability and biotransformation of cancerostatic drugs at different chloride

concentrations simulating waste water conditions. Their finding showed that oxaliplatin

degradation was increased as the concentration of chloride increases. The same study further

investigated the biotransformation potential of oxaliplatin and more than 17 reaction products

were found. Therefore, the highest removal of oxaliplatin might be attributed to its high rate

of transformation in urine that could be more adsorbed by the biomaterials than others that

are more stable like carboplatin. Conversely, high concentration of chloride in a solution

rather conserves cisplatin species and makes it stable, with an optimum concentration of 2 M

Cl-

(Nischwitz et al., 2004). Moreover, the different structure of oxaliplatin from other Pt

species also could favour for higher sorption. For instance, oxaliplatin is less polar that

60

prefers to be adsorbed in the hydrophobic structure of sorbents than staying in the solution

(Tyagi et al., 2008).

Finally, the impact of matrix effect of urine was found significant for sorption of Pt (Fig. 21).

Previous studies also confirmed that the matrix effect plays a role in sorption of precious

metals as Pt due to the interference and competition for sorption sites by presence of high

contents of organic and inorganic compounds (Salih et al., 2007).

6.6 Desorption experiment

Desorption of Pt from the biomaterials is very important given the fact that Pt is so precious

and needed to be recovered. Moreover, the biomaterials could be used repeatedly. The result

of this study revealed that 91.7% of the adsorbed Pt was recovered. A desorption experiment

conducted by Das (2010) showed that 95% of gold was recovered by using NaOH. In the

same study 90.7% of Pt(IV) desorption was obtained by using acidic thiourea solution. Zhou

et al. (2009) also found that desorption of Pt(IV) using 1 M EDTA was 92.3%. The use of

acids for desorption purposes is very common but chitosan could be easily dissolved by acids

and hence desorption was conducted by using basic solution (NaOH). Hence, the recovery

rate of Pt was found high and the regenerated media was used until three times, making its

use attractive from an environmental and economical point of view.

6.7 Economic evaluation

Design and application of any pollutant removal and recovery technology should consider

economic viability. It is one of the main factors that determine its feasibility. Economic

analysis mainly focuses on the investment and operating costs (Brunazzi et al., 2002).

The initial cost is calculated based on the selected biomaterial used for the continuous flow

system, which is activated carbon and chitosan in combination in the dual bed. First of all, the

amount of Pt discharged per hospital bed should be determined. The average Pt emission per

bed was used from results of previous studies. Kummerer (1999) measured that hospitals in

Austria, Netherlands and Germany emitted an average Pt amunt of 58.7, 22.3 and 130.4 mg

of Pt per bed per annum, respectively. The weighted average was calculated based on the

number of beds studies studied in each country and is calculated as follows.

61

yearperbedpermgxxx

5.78)18878572514(

))4.1301887()3.22857()7.582514((

We considered in our calculations a hospital having 400 beds under oncologic ward and

assumed that all beds excrete the same amount of Pt, as calculated above as the weighted

average of hospitals in three countries. Accordingly, the total emissions of Pt become 31,400

mg y-1

and 2617 mg per month. We assume to treat the urine containing this Pt amount using

the filter media tested in our study.

From the breakthrough curve drawn for cisplatin, it was concluded that saturation is reached

after percolating 600 mL of Pt solution, which is taken as the standard because cisplatin is the

most widely used CPC in hospitals. Saturation was reached after treating 60 µg of Pt and for

this 12.3 g of activated carbon and chitosan were used. Hence, 2617 mg of Pt excreted

monthly from a hospital (400 beds), requires 536 kg of biomaterials (440 kg activated carbon

and 96 kg of chitosan). The price of granular activated carbon with a description of having

higher purity (99%), large surface area and strong adsorption with high rate of regeneration

was selected one metric ton (1000 kg) costs about €579 (currency converted from $)

(Alibaba.com, 2014a). Chitosan, which is soluble in acid but insoluble in alkali and ordinary

organic solvent with 85% degree of deacetylatation costs on average €10 kg-1

(Alibaba.com,

2014b).

The total cost of the biomaterials required monthly for the dual bed column will be:

€1215))96*10()1000

579*440(( lsbiomateriaofCost

The maximum number of media regeneration will be 5 times and its cost will reduce to €243

per month. From the total 2617 mg Pt treated regeneration will be done with a recovery rate

of 91.7%, resulting in a recovery of 2400 mg. The price of Pt is currently €37.4 g-1

(Infomine,

2014). Therefore, the total amount of Pt recovered would be worth €94. The net income

generated from the recovery will be €149€243-€94 (Economically not attractive).

If we assume to use activated carbon only for treating the same amount of Pt excreted from

the above mentioned hospital beds, the price of biomaterials will drop from €1215 to €310.

Therefore, the use of aliternative low cost biomaterial is inevitable for this kind of filter

media to be economically feasible.

62

The value and quality of Pt recovered is not equal to the quality of Pt available in the market,

and only the costs for the biomaterials were taken in to account, so losses may even be higher

in reality. Therefore, we concluded that Pt recovery from cancer patient’s urine is currently

not feasible and the extraction procedure would need to be further modified to maximize its

effieicncy and capacity and/or reduce its cost.

63

7. Conclusion

Although the European Union banned the indiscriminate discharge of chemotherapy products

and their metabolites having potential carcinogenicity into the municipal waste water,

cytostatic agents and other emerging contaminants are ubiquitously found in high

concentrations in waste water coming from oncologic ward. To alleviate this problem and

comply with future rules and regulation, separate collection and treatment of cancer patient’s

urine is considered to be interesting. The main focus of the present work was to evaluate the

potential use of low cost biomaterials for the recovery of Pt from urine containing CPC.

From all biomaterials tested, activated carbon, chitosan and biochar were selected based on

their removal efficiency in the batch adsorption test. In general, the influence of pH on

sorption was found minimal and higher removal efficiency was achieved in neutral pH range,

which implies that urine may not need pH adjustment prior to the treatment. The removal of

all Pt species by sorption was found highest for activated carbon at pH 7, and decreases over

chitosan to biochar.

The amount of [PtCl6]2-

and CPC adsorbed on biomaterials increases as the concentration of

Pt in the solution increases. A matrix effect was found significant in affecting sorption of Pt

on biomaterials. On the other hand, the removal efficiency of PMO-SH and PMO-SH(50%)

was very high for all Pt species, except carboplatin during the test conducted in Milli-Q

water. The effect of pH, concentration and matrix effect were not addressed in the PMO-SH

and PMO-SH (50%) experiment.

Freundlich and Langmuir models were checked to examine the mechanism of adsorption. All

biomaterials were found to follow follow the Freundlich sorption isotherm model with strong

fitted. Fitting to the Freundlich model indicated that all biomaterials followed a multilayer

sorption process with different adsorption energy. None of them seemed to follow the

monolayer sorption model of Langmuir showing very poor fit and low correlation

coefficients. Because of this, maximum sorption capacity of the biomaterials was not

determined.

A dual bed system composed of activated carbon and chitosan was selected for further study

after finding a significant difference in sorption between the selected media and a single bed

64

containing only activated carbon. The removal efficiency of the dual bed column was

significantly higher for oxaliplatin compared to other Pt species. Saturation of this compound

was not reached for the tested volume (1 L) and further tests with higher eluent volume are

needed to assess saturation volume. The removal efficiency of this system was found to

decrease in the following order: achieved for carboplatin > [PtCl6]2-

> cisplatin.

Eventually, the conducted desorption study showed that a relatively high portion of the

temporarily adsorbed Pt could be recovered, which is very attractive to use the biomaterials

in real applications to recover this precious metal.

65

8. Further research

Since the experiment was done using synthetic urine, testing the potential of the biomaterials

for treatment of real cancer patient’s urine would be a next step. Moreover, the capacity of

biomaterials could not yet be calculated since the data did not fit to the Langmuir model,

which might be due to the fact that low concentrations were included only. Therefore, higher

concentrations may need to be included in further studies to determine sorption capacities.

Further improving the sorption efficiency of the biomaterials through different modifications

is suggested. For example, chitosan modified with thiol groups was previously tested for

Pt(IV) but not yet for other CPCs.

In the fixed bed adsorption study, the dual bed column containing activated carbon and

chitosan performed better than a single bed made from activated carbon only. It is believed

that chitosan primarily reduces the flow rate and improves the contact time between

biomaterials and Pt species, besides extraordinary removal for selected Pt species that arise

with chitosan. If its effect is only flow regulating another less expensive material can be used

instead. Mechanisms involved should be revealed. Besides that, operational factors like the

effect of bed height and diameter of the column can be further optimized for Pt removal.

Similarly, the optimum flow rate that helps to achieve maximum removal needs to be studied.

Eventually, a preliminary study on PMO-SH and PMO-SH(50%) was conducted at a specific

pH value (6), L/S ratio and Milli-Q water as aqueous medium. However, its removal

efficiency and capacity could be investigated using synthetic urine and real urine, at different

L/S ratios or sorbent doses and at a variety of pH values to determine its actual applicability.

Accordingly, further optimization of the removal efficiency of this material might be

possible.

66

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72

Annex- I: Post Hoc test result of Tukey HSD to examine differences or similarities in

sorption between biomaterials for each Pt species at pH 7; test was done after homogeneity of

variance was proven with P-value > 0.05

Dependent

Variable

(I)

biomaterial

(J)

Biomaterial

Mean

Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

[PtCl6]2-

AC Chitosan 17,00000* 1,83364 ,000 11,3739 22,6261

Biochar 62,40000* 1,83364 ,000 56,7739 68,0261

Chitosan AC -17,00000* 1,83364 ,000 -22,6261 -11,3739

Biochar 45,40000* 1,83364 ,000 39,7739 51,0261

Biochar AC -62,40000* 1,83364 ,000 -68,0261 -56,7739

Chitosan -45,40000* 1,83364 ,000 -51,0261 -39,7739

Cisplatin AC Chitosan 11,83333* 1,86130 ,002 6,1223 17,5443

Biochar 23,40000* 1,86130 ,000 17,6890 29,1110

Chitosan AC -11,83333* 1,86130 ,002 -17,5443 -6,1223

Biochar 11,56667* 1,86130 ,002 5,8557 17,2777

Biochar AC -23,40000* 1,86130 ,000 -29,1110 -17,6890

Chitosan -11,56667* 1,86130 ,002 -17,2777 -5,8557

Carboplatin AC Chitosan 10,83333 3,88463 ,071 -1,0858 22,7525

Biochar 8,66667 3,88463 ,144 -3,2525 20,5858

Chitosan AC -10,83333 3,88463 ,071 -22,7525 1,0858

Biochar -2,16667 3,88463 ,846 -14,0858 9,7525

Biochar AC -8,66667 3,88463 ,144 -20,5858 3,2525

Chitosan 2,16667 3,88463 ,846 -9,7525 14,0858

Oxaliplatin AC Chitosan 57,00000* 2,45266 ,000 49,4746 64,5254

Biochar 62,43333* 2,45266 ,000 54,9079 69,9588

Chitosan AC -57,00000* 2,45266 ,000 -64,5254 -49,4746

Biochar 5,43333 2,45266 ,147 -2,0921 12,9588

Biochar AC -62,43333* 2,45266 ,000 -69,9588 -54,9079

Chitosan -5,43333 2,45266 ,147 -12,9588 2,0921

73

Annex-II: Post Hoc test result of Tamhane to examine significant differences or similarities

in sorption of [PtCl6]2-

between tested pH levels for each biomaterial (activated carbon (AC),

chitosan and biochar); test was done after test for variance homoginity failed with P-value <

0.05

Dependent

Variable (I) pH (J) pH

Mean




AC 5,00 6,00 -11,41000 4,41623 ,936 -100,5471 77,7271

7,00 -13,23667 4,44922 ,873 -97,0898 70,6165

8,00 -13,23333 4,41989 ,878 -101,7493 75,2826

9,00 2,47000 4,42308 1,000 -85,5129 90,4529

10,00 25,94333 5,03041 ,242 -20,0589 71,9455

11,00 30,62333 4,49861 ,306 -46,4557 107,7024

6,00 5,00 11,41000 4,41623 ,936 -77,7271 100,5471

7,00 -1,82667 ,55025 ,812 -12,1054 8,4521

8,00 -1,82333 ,20661 ,113 -4,3894 ,7427

9,00 13,88000* ,26625 ,003 9,9444 17,8156

10,00 37,35333 2,41086 ,083 -11,1648 85,8715

11,00 42,03333* ,86299 ,008 25,1491 58,9176

7,00 5,00 13,23667 4,44922 ,873 -70,6165 97,0898

6,00 1,82667 ,55025 ,812 -8,4521 12,1054

8,00 ,00333 ,57895 1,000 -7,3988 7,4054

9,00 15,70667* ,60281 ,004 9,4814 21,9319

10,00 39,18000 2,47078 ,053 -1,0211 79,3811

11,00 43,86000* 1,01845 ,000 35,5582 52,1618

8,00 5,00 13,23333 4,41989 ,878 -75,2826 101,7493

6,00 1,82333 ,20661 ,113 -,7427 4,3894

7,00 -,00333 ,57895 1,000 -7,4054 7,3988

9,00 15,70333* ,32139 ,000 13,3511 18,0556

10,00 39,17667 2,41757 ,073 -8,2296 86,5829

11,00 43,85667* ,88156 ,004 29,4866 58,2267

9,00 5,00 -2,47000 4,42308 1,000 -90,4529 85,5129

6,00 -13,88000* ,26625 ,003 -17,8156 -9,9444

7,00 -15,70667* ,60281 ,004 -21,9319 -9,4814

8,00 -15,70333* ,32139 ,000 -18,0556 -13,3511

10,00 23,47333 2,42340 ,186 -23,0096 69,9563

11,00 28,15333* ,89741 ,008 15,3426 40,9640

10,00 5,00 -25,94333 5,03041 ,242 -71,9455 20,0589

6,00 -37,35333 2,41086 ,083 -85,8715 11,1648

74

7,00 -39,18000 2,47078 ,053 -79,3811 1,0211

8,00 -39,17667 2,41757 ,073 -86,5829 8,2296

9,00 -23,47333 2,42340 ,186 -69,9563 23,0096

11,00 4,68000 2,55865 ,986 -27,9253 37,2853

11,00 5,00 -30,62333 4,49861 ,306 -107,7024 46,4557

6,00 -42,03333* ,86299 ,008 -58,9176 -25,1491

7,00 -43,86000* 1,01845 ,000 -52,1618 -35,5582

8,00 -43,85667* ,88156 ,004 -58,2267 -29,4866

9,00 -28,15333* ,89741 ,008 -40,9640 -15,3426

10,00 -4,68000 2,55865 ,986 -37,2853 27,9253

Chitosan 5,00 6,00 -33,16667* 1,58044 ,008 -50,2151 -16,1182

7,00 -32,50000* 1,78481 ,002 -45,8356 -19,1644

8,00 -18,63333 1,46021 ,102 -45,2533 7,9867

9,00 -3,06667 1,76855 ,978 -16,4873 10,3539

10,00 18,03333* 2,27327 ,033 1,9944 34,0723

11,00 18,60000* 1,57021 ,043 1,1069 36,0931

6,00 5,00 33,16667* 1,58044 ,008 16,1182 50,2151

7,00 ,66667 1,23063 1,000 -9,6214 10,9548

8,00 14,53333* ,67905 ,016 5,5917 23,4750

9,00 30,10000* 1,20692 ,001 20,2065 39,9935

10,00 51,20000* 1,86994 ,007 27,7944 74,6056

11,00 51,76667* ,89132 ,000 45,6936 57,8397

7,00 5,00 32,50000* 1,78481 ,002 19,1644 45,8356

6,00 -,66667 1,23063 1,000 -10,9548 9,6214

8,00 13,86667 1,07186 ,086 -4,1070 31,8404

9,00 29,43333* 1,46439 ,001 19,4645 39,4022

10,00 50,53333* 2,04559 ,002 33,1096 67,9571

11,00 51,10000* 1,21747 ,000 40,6054 61,5946

8,00 5,00 18,63333 1,46021 ,102 -7,9867 45,2533

6,00 -14,53333* ,67905 ,016 -23,4750 -5,5917

7,00 -13,86667 1,07186 ,086 -31,8404 4,1070

9,00 15,56667 1,04456 ,063 -1,7867 32,9200

10,00 36,66667* 1,76949 ,041 3,3515 69,9819

11,00 37,23333* ,65490 ,001 28,8423 45,6244

9,00 5,00 3,06667 1,76855 ,978 -10,3539 16,4873

6,00 -30,10000* 1,20692 ,001 -39,9935 -20,2065

7,00 -29,43333* 1,46439 ,001 -39,4022 -19,4645

8,00 -15,56667 1,04456 ,063 -32,9200 1,7867

10,00 21,10000* 2,03142 ,029 3,4359 38,7641

11,00 21,66667* 1,19350 ,004 11,5870 31,7463

75

10,00 5,00 -18,03333* 2,27327 ,033 -34,0723 -1,9944

6,00 -51,20000* 1,86994 ,007 -74,6056 -27,7944

7,00 -50,53333* 2,04559 ,002 -67,9571 -33,1096

8,00 -36,66667* 1,76949 ,041 -69,9819 -3,3515

9,00 -21,10000* 2,03142 ,029 -38,7641 -3,4359

11,00 ,56667 1,86130 1,000 -23,3975 24,5308

11,00 5,00 -18,60000* 1,57021 ,043 -36,0931 -1,1069

6,00 -51,76667* ,89132 ,000 -57,8397 -45,6936

7,00 -51,10000* 1,21747 ,000 -61,5946 -40,6054

8,00 -37,23333* ,65490 ,001 -45,6244 -28,8423

9,00 -21,66667* 1,19350 ,004 -31,7463 -11,5870

10,00 -,56667 1,86130 1,000 -24,5308 23,3975

Biochar 5,00 6,00 7,40000 2,01550 ,484 -10,0606 24,8606

7,00 -3,33333 1,29915 ,758 -12,6230 5,9564

8,00 3,23333 1,09494 ,786 -9,6421 16,1087

9,00 7,56667 1,35565 ,105 -1,7985 16,9318

10,00 ,56667 1,53876 1,000 -10,0647 11,1980

11,00 4,36667 1,07600 ,562 -9,6567 18,3901

6,00 5,00 -7,40000 2,01550 ,484 -24,8606 10,0606

7,00 -10,73333 1,91862 ,244 -30,7365 9,2698

8,00 -4,16667 1,78668 ,949 -32,9061 24,5727

9,00 ,16667 1,95732 1,000 -18,5901 18,9235

10,00 -6,83333 2,08833 ,554 -23,3348 9,6681

11,00 -3,03333 1,77514 ,995 -33,1119 27,0452

7,00 5,00 3,33333 1,29915 ,758 -5,9564 12,6230

6,00 10,73333 1,91862 ,244 -9,2698 30,7365

8,00 6,56667 ,90431 ,114 -2,2877 15,4210

9,00 10,90000* 1,20692 ,018 2,6045 19,1955

10,00 3,90000 1,40949 ,709 -6,8643 14,6643

11,00 7,70000 ,88129 ,091 -1,9948 17,3948

8,00 5,00 -3,23333 1,09494 ,786 -16,1087 9,6421

6,00 4,16667 1,78668 ,949 -24,5727 32,9061

7,00 -6,56667 ,90431 ,114 -15,4210 2,2877

9,00 4,33333 ,98376 ,416 -6,1439 14,8106

10,00 -2,66667 1,22384 ,953 -18,4353 13,1019

11,00 1,13333 ,53645 ,899 -2,5892 4,8559

9,00 5,00 -7,56667 1,35565 ,105 -16,9318 1,7985

6,00 -,16667 1,95732 1,000 -18,9235 18,5901

7,00 -10,90000* 1,20692 ,018 -19,1955 -2,6045

76

8,00 -4,33333 ,98376 ,416 -14,8106 6,1439

10,00 -7,00000 1,46173 ,192 -17,5616 3,5616

11,00 -3,20000 ,96264 ,697 -14,6688 8,2688

10,00 5,00 -,56667 1,53876 1,000 -11,1980 10,0647

6,00 6,83333 2,08833 ,554 -9,6681 23,3348

7,00 -3,90000 1,40949 ,709 -14,6643 6,8643

8,00 2,66667 1,22384 ,953 -13,1019 18,4353

9,00 7,00000 1,46173 ,192 -3,5616 17,5616

11,00 3,80000 1,20692 ,785 -13,2328 20,8328

11,00 5,00 -4,36667 1,07600 ,562 -18,3901 9,6567

6,00 3,03333 1,77514 ,995 -27,0452 33,1119

7,00 -7,70000 ,88129 ,091 -17,3948 1,9948

8,00 -1,13333 ,53645 ,899 -4,8559 2,5892

9,00 3,20000 ,96264 ,697 -8,2688 14,6688

10,00 -3,80000 1,20692 ,785 -20,8328 13,2328

*. The mean difference is significant at the 0.05 level.

77

Annex-III: Post Hoc test result of Tamhane to examine differences or similarities in sorption

of cisplatin between tested pH levels for each biomaterial (activated carbon (AC), chitosan

and biochar); test was done after test for variance homogeneity failed with P-value < 0.05

Dependent

Variable (I) pH (J) pH

Mean




AC 5,00 7,00 -10,66667 2,98142 ,142 -25,5754 4,2421

9,00 -16,00000* 2,21108 ,020 -28,0486 -3,9514

11,00 -15,00000 3,39935 ,093 -33,4648 3,4648

7,00 5,00 10,66667 2,98142 ,142 -4,2421 25,5754

9,00 -5,33333 2,62467 ,582 -21,6147 10,9481

11,00 -4,33333 3,68179 ,889 -22,5862 13,9195

9,00 5,00 16,00000* 2,21108 ,020 3,9514 28,0486

7,00 5,33333 2,62467 ,582 -10,9481 21,6147

11,00 1,00000 3,09121 1,000 -20,5872 22,5872

11,00 5,00 15,00000 3,39935 ,093 -3,4648 33,4648

7,00 4,33333 3,68179 ,889 -13,9195 22,5862

9,00 -1,00000 3,09121 1,000 -22,5872 20,5872

Chitosan 5,00 7,00 ,66667 7,24952 1,000 -55,2431 56,5765

9,00 -3,00000 7,06321 ,999 -65,1438 59,1438

11,00 20,66667 7,31817 ,392 -33,4707 74,8040

7,00 5,00 -,66667 7,24952 1,000 -56,5765 55,2431

9,00 -3,66667 2,98142 ,874 -19,0092 11,6759

11,00 20,00000* 3,54338 ,029 2,8372 37,1628

9,00 5,00 3,00000 7,06321 ,999 -59,1438 65,1438

7,00 3,66667 2,98142 ,874 -11,6759 19,0092

11,00 23,66667* 3,14466 ,016 6,9077 40,4256

11,00 5,00 -20,66667 7,31817 ,392 -74,8040 33,4707

7,00 -20,00000* 3,54338 ,029 -37,1628 -2,8372

9,00 -23,66667* 3,14466 ,016 -40,4256 -6,9077

Biochar 5,00 7,00 6,66667 3,72678 ,710 -20,8061 34,1395

9,00 12,33333 4,25572 ,396 -21,5164 46,1830

11,00 11,00000 7,08676 ,826 -56,7605 78,7605

7,00 5,00 -6,66667 3,72678 ,710 -34,1395 20,8061

9,00 5,66667 5,33333 ,924 -20,4583 31,7917

11,00 4,33333 7,78175 ,997 -44,8077 53,4744

9,00 5,00 -12,33333 4,25572 ,396 -46,1830 21,5164

7,00 -5,66667 5,33333 ,924 -31,7917 20,4583

11,00 -1,33333 8,04846 1,000 -47,8637 45,1971

11,00 5,00 -11,00000 7,08676 ,826 -78,7605 56,7605

78

7,00 -4,33333 7,78175 ,997 -53,4744 44,8077

9,00 1,33333 8,04846 1,000 -45,1971 47,8637

*. The mean difference is significant at the 0.05 level.

79

Annex-IV- Post Hoc test result of Tukey HSD to compare differences or similarities in

sorption between different Pt species by using dual bed column packed with activated carbon

and chitosan; test was done after homogeneity of variance was proven with P-value > 0.05

(I) Pt. Species (J) Pt. Secies

Mean




[PtCl6]2-

Cisplatin 9,40000 6,34354 0.453 -7,2632 26,0632

Carboplatin -8,75000 6,34354 0.516 -25,4132 7,9132

Oxaliplatin -55,30000* 6,34354 < 0.001 -71,9632 -38,6368

Cisplatin [PtCl6]2- -9,40000 6,34354 0.453 -26,0632 7,2632

Carboplatin -18,15000* 6,34354 0.027 -34,8132 -1,4868

Oxaliplatin -64,70000* 6,34354 < 0.001 -81,3632 -48,0368

Carboplatin [PtCl6]2- 8,75000 6,34354 0.516 -7,9132 25,4132

Cisplatin 18,15000* 6,34354 0.027 1,4868 34,8132

Oxaliplatin -46,55000* 6,34354 < 0.001 -63,2132 -29,8868

Oxaliplatin [PtCl6]2- 55,30000* 6,34354 < 0.001 38,6368 71,9632

Cisplatin 64,70000* 6,34354 < 0.001 48,0368 81,3632

Carboplatin 46,55000* 6,34354 < 0.001 29,8868 63,2132

applicability of biomaterials for the recovery of platinum...

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