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Design of potent fluoro-substituted chalcones as antimicrobial agents

Abstract Owing to ever-increasing bacterial and fungal drug resistance, we attempted to develop novel antitubercular and antimicrobial agents. For this purpose, we developed some new fluorine-substituted chalcone analogs (3, 4, 9–15, and 20–23) using a structure–activity relationship approach. Target compounds were evaluated for their antitubercular efficacy against Mycobacterium tuberculosis H37Rv and antimicrobial activity against five common pathogenic bacterial and three common fungal strains. Three derivatives (3, 9, and 10) displayed significant antitubercular activity with IC50 values of ≤16,760. Compounds derived from trimethoxy substituent scaffolds with monofluoro substitution on the B ring of the chalcone structure exhibited superior inhibition activity compared to corresponding hydroxy analogs. In terms of antimicrobial activity, most compounds (3, 9, 12–14, and 23) exhibited moderate to potent activity against the bacteria, and the antifungal activities of compounds 3, 13, 15, 20, and 22 were comparable to those of reference drugs ampicillin and fluconazole.

Introduction
Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most important diseases worldwide. According to a 2014 report by the WHO, incidences, prevalence, and mortality rates of tuberculosis have globally decreased. However, in 2014 alone, an estimated 9.6 million new cases of tuberculosis and 1.5 million deaths caused by the disease were reported 1 . Furthermore, the emergence of multidrug-resistant and extensively drug-resistant strains has led to a growing need for effective novel agents in the fight against tuberculosis. Unfortunately, there are very few new drugs being developed for this purpose 2 . Consequently, there is a growing need for the development of effective drugs to combat tuberculosis.
The incidences of failure in the treatment of bacterial and fungal infections have increased because of the emergence of multidrug-resistant strains due to misuse of antimicrobial drugs 3 . Therefore, the synthesis of effective, novel antimicrobial compounds has become extremely important.
Chalcones are compounds with simple chemistry that offer easy synthetic access to various substituted derivatives. As well as being important constituents of various natural products, chalcones, also called a,b-unsaturated ketones, are important synthetic precursors. Chalcones and their synthetic derivatives possess extensive pharmacological properties, such as antihypertensive, antiplatelet, antidiabetic, antineoplastic, antiangiogenic, antiretroviral, anti-inflammatory, antihistaminic, antioxidant, antitubercular, antifungal, anti-invasive, and antiulcer properties 4 . Chalcones can be easily synthesized via the Claisen-Schmidt reaction of acetophenones and benzaldehydes under basic conditions.
In recent years, compounds containing fluorine have become common as potential lead drugs 5 . It has been reported that insertion of a fluorine atom into a biologically active compound results in minimal steric change, thus maintaining interactions with enzyme active sites, receptor recognitions sites, and other biological systems 6 . Furthermore, it has been indicated that the high electronegativity of fluorine can lead to significant changes in the physical and chemical properties of the molecule 6,7 . For example, it is known that a moderate to high change in the lipophilicity of a compound can impart it with improved antitubercular activity. It has been established in recent literature that fluorine-substituted compounds exhibit improved bioactivity and efficacy. Compounds with a chalcone backbone have also been reported to exhibit antimicrobial activity [8][9][10] .
In this study, we performed antitubercular and antimicrobial activity studies on eleven B-ring fluoro-substituted chalcones and two nonsubstituted chalcones that we have previously synthesized in an efficient, high-yielding manner 11 . These activity studies indicated that they may be used in the treatment of tuberculosis and other bacterial and fungal infections.

General
All reagents used were commercially available unless otherwise specified and all solvents were distilled before use. Melting points were measured with Gallenkamp melting point devices. IR Spectra: PerkinElmer Spectrum One FT-IR spectrometer. 1 H-and 13 C-NMR Spectra: Varian 400 and Bruker 400 spectrometers. Elemental analysis results were obtained on a Leco CHNS-932 instrument.

Antitubercular studies
Agar proportion method: The MIC values of each synthesized compound 3, 4, 9-15, 20-23 were obtained by agar dilution in duplicate as recommended by the Clinical Laboratory Standards Institute (CLSI) 12,13 . Positive and negative growth controls were used in each assay. INH (Sigma I3377) and EMB (Sigma E4630) were used as control agents. M. tuberculosis H37Rv was used as the standard strain and was provided by Refik Saydam National Public Health Agency, National Tuberculosis Reference Laboratory, Ankara, Turkey. Stock solutions of the tested compounds and reference compounds were prepared in DMSO/H 2 O (50%) at a concentration of 1000 mg/mL. These solutions were then filtered through a 0.22mm membrane filter (Millex-GP SLGP033RS, Merck Millipore, Merck KGaA Darmstadt, Germany). Middlebrook 7H10 agar medium (BBL, Becton Dickinson and Company, Sparks, MD) was supplemented with oleic acid-albumin-dextrose-catalase (OADC, BBL, Becton Dickinson and Company, Sparks, MD). Compounds and control agents were added to obtain an appropriate final concentration in the medium. The final concentrations of INH and EMB were 0.2-1 mg/mL and 5-10 mg/mL, respectively. Compounds were prepared at final concentrations of 5, 10, 20, 40, and 80 mg/mL. Agar without any reference or tested compounds was used as a positive growth control, and 3.0 mL of prepared medium was dispensed into sterile tubes. The DMSO concentration in the final solutions was not above 1% for antimycobacterial activity.
Inoculum preparation: H37Rv was maintained in Lowenstein-Jensen medium. A culture suspension was prepared by subculturing in Middlebrook 7H9 broth (BBL, Becton Dickinson and Company, Sparks, MD) supplemented with 10% OADC at 37 C for 7-10 days, until a density corresponding to 10 -2 to 10 -4 dilutions were obtained from McFarland standard No. 1. Then, 0.1 mL of the diluted suspension was inoculated onto the control and the other tubes with compounds in different concentrations. The tubes were incubated at 37 C in a 5% CO 2 atmosphere for 3 weeks. The MIC values were defined as the lowest concentration that inhibited more than 90% of the bacterial growth, and the results for INH and EMB were interpreted according to the CLSI. The MIC was considered as the lowest concentration that showed no visible colonies in all dilutions. The biological activity studies of the compounds were performed twice. The results of these two studies were almost the same. The IC 50 values were calculated as the average activity values for the compounds. The results are given in Figure 1.
Antimicrobial activity studies Antimicrobial susceptibility testing was performed by the modification of literature methods 14,15 . We used the microbial strains The fungal and bacterial cell inoculum were prepared from a stock culture grown in tryptic soy agar (TSA) at 28 C for 24 h, and Mueller-Hinton agar (MHA) at 37 C for 24 h, respectively. The microorganism suspension concentrations were adjusted according to McFarland 0.5 turbidity tubes using sterilized saline. Stock solutions of the title compounds were prepared in DMSO at 1000 mg/ mL. A modified microdilution test was applied for antimicrobial activity, and the experiments were run in duplicate independently.
For antifungal activity testing, 100 mL Tryptic Soy Broth (TSB) was added to each of the 11 wells. A 100 mL aliquot of the tested chemical solution was added to the first well, and twofold dilutions were prepared. Then, 5 mL of fungal suspension was added to each tube except the last one, which acted as the control well.
For antibacterial activity testing, 100 mL Mueller-Hinton broth (MHB) was added to each of the 11 wells. A 100 mL aliquot of the chemical derivative solution was added to the first tube, and twofold dilutions were prepared. Then, 5 mL of the bacterial suspension was added to each tube, except the last control well. A control tube containing 5 mL of the fungal and bacterial suspensions alone without the tested compounds was also prepared. All plates were incubated at 28 C (for fungi) and at 37 C (for bacteria) for 24 h. After incubation, the MICs (Tables 2 and 3) were obtained by noting the growth inhibitions. The concentration resulting in a 50% reduction in the optical density (OD) values was compared to a reproduction control at 450 nm by spectrophotometric evaluation and defined as the MIC value. Fluconazole and ampicillin were used as reference drugs. The results were read visually and by measuring optical density for 24 h.

Chemistry
We previously reported the synthesis of 11 B-ring fluoro-substituted and two nonsubstituted chalcone derivatives 11 . The general structure of synthesized compounds is shown in Scheme 1.
Using this procedure 11 , base-catalyzed Claisen-Schmidt condensation afforded chalcones 3, 9-11, and 12 in yields of 80-95%. Hydrogenation of the chalcone compounds over Pd-C gave 4, 13, 14, and 15 in yields of 70-90%. Trihydroxy-chalcones were synthesized using the same procedure. Before the condensation reaction, the methoxymethyl ether (MOM) group was used to protect the hydroxyl groups. The base-catalyzed reaction was then used to afford MOM-protected chalcones. After deprotection, hydrogenation of 20 and 21 over Pd-C gave the target compounds 22 and 23 in yields of 80% and 85%, respectively. The general method for the synthesis of these chalcones is briefly described in Scheme 2. All compounds except 11 were previously synthesized by our group 11 . We report here the synthesis of compound 11 and its structural details.
Modification of compound 3 (the methoxy derivative with non fluoro substituent) for activity modulation was made through preparation of compounds with fluoro substituents on the B ring only (9, 10, 11, and 12), and with hydroxyl substituents on the A ring and fluoro substituents on the B ring (20 and 21). Versions of these compounds with semisaturated or saturated linkers were also prepared (4, 13-15, 22, and 23). The synthesized compounds were fully characterized with common spectroscopy techniques.
To evaluate the potential of the basic pharmacophore of chalcone, 3 was evaluated for its antimicrobial efficacy against the Mycobacterium tuberculosis H37Rv strain and several other bacteria and fungi. The different functional groups carry on aromatic rings of chalcones are responsible for their antimicrobial activities. The details of the substitution patterns in the target compounds are presented in Table 1.

Antitubercular studies
All the target compounds (3,4,(9)(10)(11)(12)(13)(14)(15), and 23) were screened against M. tuberculosis H37Rv using the agar proportion method. Isoniazid (INH) and ethambutol (EMB) were used as the positive drug standards for the assay. The in vitro antimycobacterial activity in terms of the minimum inhibitory concentration (MIC) values of the target compounds and the standard drugs are shown in Figure 1. The three derivatives 3, 9, and 10 show significant activities, with IC 50 values 16,760, and compounds 11 and 12 exhibit moderate activities with IC 50 values 31.63. The remaining eight compounds 4, 13-15, 20-22, and 23 exhibit very low activity. The compounds derived from trimethoxy and non-or monofluorosubstituted chalcone scaffolds exhibit superior inhibition activity to their hydroxyl-and difluoro-substituted analogs. This observation clearly indicates that the trimethoxy A ring and fluoro groups on the B ring enhance the inhibition activity of the molecules. Furthermore, these results suggest that further structural modification of the above-mentioned drug molecules (3, 9, and 10) using a structure-activity relationship (SAR) approach could be a promising strategy for the identification of new leads toward the development of potent antitubercular agents.

Antibacterial studies
The antibacterial activities of the target compounds (3,4,(9)(10)(11)(12)(13)(14)(15), and 20-23) against the five common pathogenic bacterial strains S. aureus, S. pyogenes, E. faecalis, E. coli, and P. aeruginosa is shown in Table 2. The experiments were performed using the microdilution method with reference to the MIC values of the compounds. The well-known commercial antibiotic ampicillin was used as the standard drug during the assay.
Compounds 3 and 4 show significant activity against almost all the bacterial strains, and compound 23 is the most active compound against S. aureus. Interestingly, all the compounds derived from trimethoxy-substituted chalcone scaffolds exhibit significant inhibition activities against all the tested strains, while the other compounds exhibit moderate and low activity.

Antifungal studies
Compounds 3, 4, 9-15, and 20-23 were evaluated for antifungal activity against the C. albicans, C. glabrata, and C. parapsilosis fungal strains. The MIC values obtained for the compounds are given in Table 3. Fluconazole was used as the reference for inhibitory activity against fungi. The antifungal activities of all the compounds are comparable to that of fluconazole. Compounds 3,13,15,20, and 21 are the most active compounds, with compounds 3 and 20 exhibiting excellent activity against C. albicans at MICs of 15.62 mg/mL. Compounds 3, 13, 15, and 21 exhibit very good activity against C. glabrata at an MIC of 31.25 mg/mL, while the other compounds (9-13, 20, 22, and 23) exhibit moderated inhibition with MICs of 62.5 and 125 mg/mL, as compared to the standard fluconazole (31.25 mg/mL). Table 1. Substitution patterns and antitubercular activity of target compounds (3, 4, 9-15, and 20-23 Compounds 13, 15, and 21 exhibit the highest activity against P. parapsilosis, with MICs of 31.25 mg/mL, whereas all other compounds exhibit low inhibitory activity compared to that of fluconazole, with MICs of 62.5 mg/mL. Compound 21, the hydroxyl and difluoro-substituted chalcone analog, exhibits the highest activity.

SAR
The SAR of compounds 3, 4, 9-15, and 20-23 was explored using the data presented in Tables 1-3, and reveals that the presence of three methoxy groups on the A ring of the chalcone increases antitubercular activity and that activity decreases in the presence of hydroxyl groups on the A ring. Chalcone and saturated chalcone compounds exhibit more pronounced antibacterial activity.
More specifically, compounds with fluoro substituents in position 2 and/or position 5 of the B ring in the chalcone structure exhibit significantly increased potency against the microbial strains. The influence of Log p on the antifungal activity of the compounds appears to be important: as Log p increases, the antifungal activity increases. Furthermore, the use of semi-saturated linker compounds appears to improve activity against all targets.

Conclusions
Recently, chalcone-like compounds, such as licochalcone A, which are present in Glycyrrhiza inflata, and 24 and 25, present in Piper sanctum, have been reported to exhibit potent antitubercular activity against Mycobacterium tuberculosis, with MICs of 7.1, 32, and 4.0 mg/mL, respectively (Figure 2) 16,17 .
In this study, based on the recent literature data, we designed and tested a series of 2,4,6-trimethoxy or hydroxyl and non/monoor difluoro-substituted chalcone derivatives in order to improve the antitubercular and antimicrobial activity of previously synthesized chalcones and to gain an understanding of their SAR in the context of anti-M. tuberculosis and antimicrobial activities (Tables 1-3).
Preliminary studies revealed that compounds (3,9,10) exhibited the greatest antitubercular activity with IC 50 values 16,760, which are comparable to the antitubercular drugs INH and EMB used as controls.
Modification of compound 3 for activity modulation was made by introducing fluorine atoms or saturation. These compounds were found to be either far less active than 3 or inactive. It is widely known that molecular flexibility plays an important role in drug-protein interactions. However, we found that the conformationally restricted chalcones were more active than the other chalcones. This modification was made in such a way that the total lipophilic nature of the compounds was similar (The Log p values for compounds are shown in Table 1). We also attempted to assess the importance of the structure of the chalcone compounds to their effectiveness as antimicrobial agents.
The synthesized compounds were also evaluated for their in vitro antimicrobial activity against several bacterial and fungal strains. The tested compounds were inactive against bacteria and fungi, and they did not exhibit significant antibacterial and antifungal activities, except against S. aureus, in comparison to ampicillin and fluconazole. However, the chalcone analogs exhibited considerable antimicrobial activity. Interestingly, compounds 3 and 4 exhibited wider activity than the other compounds. Compounds 3, 20, and 11, 23 exhibited higher antibacterial and antifungal activity against S. aureus and C. albicans, respectively.
In conclusion, assessment of the biological activity of these molecules indicated that conformationally restricted chalcones are superior to their straight chain analogs, possibly due to molecular   flexibility, and may be good leads for the future development of antitubercular drugs.


Skeletal dysmorphology in fetus following colloidal silver ingestion in pregnant Swiss albino mice

The present industrial sector related to textile industry, medicine product industry, house hold industry have enormously spread the use of Silver nanoparticle. Varieties of teratogenic effects are executed due to silver nanoparticle among which one of the important is skeletal dysmorphology. The discovery of the new facts can be compared with the quotes from some studies1 reveals that there is some chemical agents and drugs can induce teratogenic effects on the contrary silver nanoparticle induces skeletal dysmorphology in higher dose oral gavages schedules. On the due course of development it evokes major congenital abnormalities of various systems of offspring’s which is seen in developed countries like USA and Canada about 3 to 5% and in developing countries like India, Mauritania etc about 20 to 25%.2 The devastating effects of drug, manually made colloidal solution, viruses and other facts related to our ecology is responsible for 1% of congenital defects of known and unknown etiology.3 Old studies on invitro risk assessment reported silver nanoparticle translocation which causes decreased cell viability, cell membrane damage and other histological hazardous effects. Several other studies also indicated silver nanoparticle to be most toxic among all metal nanoparticles.4‒6 A toxicity of minimum level is denoted when HL-60 cell grown on metal silver plate. Some researchers did accumulation of nano silver on cells experiment7‒11 and denoted that maximum accumulation can be cytotoxic in rat liver cell and Mammalian germ line stem cells.12

Introduction
The present industrial sector related to textile industry, medicine product industry, house hold industry have enormously spread the use of Silver nanoparticle. Varieties of teratogenic effects are executed due to silver nanoparticle among which one of the important is skeletal dysmorphology. The discovery of the new facts can be compared with the quotes from some studies 1 reveals that there is some chemical agents and drugs can induce teratogenic effects on the contrary silver nanoparticle induces skeletal dysmorphology in higher dose oral gavages schedules. On the due course of development it evokes major congenital abnormalities of various systems of offspring's which is seen in developed countries like USA and Canada about 3 to 5% and in developing countries like India, Mauritania etc about 20 to 25%. 2 The devastating effects of drug, manually made colloidal solution, viruses and other facts related to our ecology is responsible for 1% of congenital defects of known and unknown etiology. 3 Old studies on invitro risk assessment reported silver nanoparticle translocation which causes decreased cell viability, cell membrane damage and other histological hazardous effects. Several other studies also indicated silver nanoparticle to be most toxic among all metal nanoparticles. [4][5][6] A toxicity of minimum level is denoted when HL-60 cell grown on metal silver plate. Some researchers did accumulation of nano silver on cells experiment [7][8][9][10][11] and denoted that maximum accumulation can be cytotoxic in rat liver cell and Mammalian germ line stem cells. 12 The current study also enlights the present world about effect of silver nanoparticle on skeletal system. It was undertaken to evaluate & assess the teratogenic effects: skeletal dysmorphology of Swiss albino mice fetuses through repeated oral gavages.

Material and methods
The present study was carried out, with rules and regulation for care and use of laboratory animals of the animal house and the respective experiment were approved by Central Animal Ethical Committee. 25 female Swiss albino mice with an average age 45days and 25 to 35gm weight were chosen randomly from Animal house, from different breeding colonies. These female non pregnant adults were intentionally copulated by male mouse of same variety with an aim for procuring 10 pups of further generation. In the plastic polycarbonate cage with stainless steel toppings single strong and stout heterozygous male were placed with single female in an atmosphere continuously maintained at 20-25˚C and 55±5% humidity, 12hrs subsequently light and dark cycle. The female mice were examined for vaginal cross plug and presence of well visualize plug were marked as ascertained pregnancy and assigned day zero of gestational. On day 7, 8 and 9 of gestation plugged females were chequed for pregnancy by abdominal palpation.

Characterization of AgNps
Silver nanoparticles from research purchasing sources were evaluated for their primary size by TEM and for their agglomeration in cell medium by photon cross correlation spectroscopy (PCCS) and ultraviolet-visible (UV-Vis) spectroscopy, Dynamic light Scattering and Zeta potential. Representative TEM pictures of the AgNps were showed. The TEM images of the PVP coated AgNps dispersed and suspended in 1.5M NaCl confirmed the primary particle size stated by the manufacturers. The uncoated silver nanoparticles found heterogeneous distribution within the range of 50 to 100 nm, most of them being around 71nm.
In the case of suspended powder AgNps, TEM images showed that in an aqueous environment the silver nanoparticles clumping and form large aggregates, many of which are larger than 50nm. This result is in consistent with the results of hydrodynamic size distribution which was obtained from the dynamic light scattering method (zeta sizer). EDX analyses were confirmed that only elemental silver was presented in both colloidal and dry powdered AgNps. According to the ICP-AES results, the concentrations of Ag ions in the acid digested stocks of colloidal and suspended powder AgNps were 1908.1 and 447.2mgl-1 respectively. Also results of the XRD pattern confirms the crystallinity of powdered type AgNps and presence of elemental crystalline silver, also other phases, except metallic silver, were not present in the sample. In addition the XRF results showed that purity of powdered type AgNps was 97.86%.

Exposure of AgNps colloidal solution through repeated oral gavages
Pregnant mice were administered double with normal saline (Sham control) as vehicle and in a dose of 5, 10, 15, 20mg/kg b.w. were given through repeated oral gavages, from 7 to 12 gestational day with the help of a blunt tip curved shaft feeding needle and van attached. Sham control animals are included in experiment with treated animals and untreated animals.

Dissection and evisceration
On GD 18, right and left horns of uterus of pregnant mother were exteriorized following ventral laparotomy under deep ether/chloroform anesthesia and 250 fresh collected dams through dissection were washed with tap water on Petri dish to remove blood and amniotic fluid and dried by filter paper and dehydrated in 95% alcohol for 48hrs at last exposed to 1% KOH for consistence evisceration and Alizarin staining protocol for proper visualization of bones either normal or abnormal. 13 Method (Assessment of skeletal dysmorphology by the technique of evisceration and Alizarin staining)-: Skeletal dysmorphology study was done by the technique of evisceration and Alizarin staining. All specimens (50 pups) from each group were dehydrated in 95% of alcohol ( rectified spirit) for 48 hours and then removed and washed by running tap water and then eviscerated by 1% KOH solution (KOH 1gm+100ml d.w.). After doing ventral laparotomy vital organs are removed. The specimens were dipped thoroughly in plenty of 1% potassium hydroxide (KOH) solution for at least 2 to 4days until the flesh, tendon, muscles and viscera's were transparent & clear and disband followed by crystal clear visibility of the bones and cartilages. Every day solution was changed when it become discolored and wastes.
The crystal clear bone and cartilage visible specimen were then placed in same 1% KOH and then 6 to 8 drops of 1% Alizarin stain was added to color it deep purplish red. The solution was gently shaked a few times per hour. Then the same procedure was continued for another 2-3days in order to give deep purplish red color to bone and cartilages. For removal of any over staining and smoothening of the samples, the same are then passed through 40%, 80% (40ml glycerol+60ml distilled water, 80ml glycerol+20ml distilled water) and 1 to 2 crystal thymol added for long term preservation finally maintained in 100% glycerol, served for photograph & powerful hand lens observation.
In total 250 fetuses were collected from 25 mothers from 1, 2, 3, 4 and 5 groups out of which all samples were exposed to skeletal dysmorphology experimentation. 60 fetuses were observed resorption.

Statistics
Percentage of skeletal malformation was evaluated from each group and assessed by number of bone involved in the dysmorphology and finally the percentages were compared between the group and within the group for dose dependant teratogenicity factor. Multi ANOVA and Descriptive statistics (post hoc analysis) test used for this purpose.

Observation and findings
The percentage of skeletal dysmorphology was found higher and highest in 15mg AgNps and 20mg AgNps/ kg b.w. treated group pups ( Table 1).
The lower dose AgNps treated groups (0.5 & 1mg/kg b.w.) were observed very minimal percentages of skeletal anomalies so these treated groups were almost considered as control group fetuses (Figure1). One of the 5mg AgNps/ kg b.w. treated group pup demonstrated unossified 5th sternal segment from the sternal bar and unossified Manumbrium segment which is diagnosed as sternal cleft. Other fetuses demonstrated maxillary and mandibular hypoplasia, unossified frontal, parietal and nasal bone, failure of fusion of posterior segment of the hip bone, 10,11 and 12th rib seen rudimentary in left side of one of the fetus. Absence of mid shaft of 6 to 12th rib along with increase of the space between successive ribs were denoted in multiple fetuses from different groups and diagnosed as increased web space, Absence of body & spinous process is seen from whole thoracic vertebral segments which is diagnosed as neural tube defect of close type in 1% cases from this treated group fetuses. The percentage of skeletal dysmorphology calculated in this group was 32% ( Figure 2).  The 10mg AgNps treated group fetuses demonstrated skeletal dysmorphology in the form of absence of lumbar and sacral vertebra, absence of hip bones, severely degenerated ribs without cartilages, frontal and parietal bone showed unossified and undeveloped, scoliosis is observed in thoracic and lumbar region in one of the fetus, absence of cervical vertebra is seen, maxillary hypoplasia, disclosure of sagittal and coronal sutures are also seen. Undeveloped lower limb bones are seen on both sides of one of the fetus. The percentage of skeletal dysmorphology calculated in this group was 44% (Figure 3). Among 15mg AgNps treated group 8 pups were observed absence of cervical vertebrae with absence of 12th rib and rest of the vertebrae were observed undeveloped spinous process, Metatarsal cleft is also seen on dorsal view of foot of one of the fetus, unossified hip bone on ventral view with absence of segment & fibrosis is also seen, unossified temporal bone which is found fibrosed on lateral view of skull, absence of proximal segment of innominate bone, vertebral column anomaly was seen with rudimentary 6th and 9th rib and absence of 10th,11th and 12th is observed ribs in one of the fetus, unossified parietal bone was also seen, Anencephaly with scoliosis vertebrae was seen in L1-L5 in one of the fetus, unossified hip bone was seen, unossified scapulae of both side was seen on dorsal view of one of the fetus, Absence of cartilage was seen from 6th to 12th rib in one of the fetus. The percentage of skeletal dysmorphology calculated in this group was 60% (Figure 4).

Figure 4 15mg AgNps treated group fetus Alizarin stain dorsal view (A, B) and ventral view (C)
The 20mg/ kg b.w. AgNps treated group fetuses were presented highest percentage of skeletal dysmorphology (76%). Scoliosis was seen in lumbar region in one of the fetus, one fetus demonstrated unossified occipital bone, unossified scapulae and hip bone, big fibrosis is observed in maxilla, frontal and parietal bones, same is also observed in second to sixth segment of sternum., cleft is observed in the ankle joint, increased space is observed between 1st and 2nd rib and 5th to 8th ribs of left side of one of the fetus, also cleft is observed in symphisis pubis joint of one of the fetus , unossified mandibular hypoplasia, absence of spinous process with wavy ribs is also observed, in one fetus we observed complete fusion of the 2nd to 8th cervical vertebrae which is clearly differentiated from rest of the 1st cervical vertebrae. We also observed mild scoliosis and unossified occipital and parietal bone and absence of scapulae on right side of one of the fetus, hypo plastic vertebrae is also seen ( Figure 5).

Discussion
The present work was undertaken to study the effect of prenatal exposure of silver nanoparticle colloidal solution through repeated oral gavages on developing skeleton of mice embryos. The skeletal dysmorphology met in this study were in the form of unossified temporal, frontal and occipital bones. As revealed by the groups 5mg,10mg, 15mg, 20mg/kg b.w. maxillary and mandibular hypoplasia, nasal bone hypoplasia, scoliosis, cleft, absence of segments of hip bone, failure of fusion of segments and large cases of increased intercostal spaces and other rib anomalies are encountered during the course of practical work conducted in this present study. Alizarin stain completely failed to reach up to tail region to stain the tail. There are plenty of data available among nanosilver toxicity from various manufactured products, this existing study indicated dysmorphology of skeletal system of vertebrate pups of Swiss albino mouse variety which is predominant among several type of teratogenicities executed till today (Figure 6). Nanosilver shows antagonistic properties in lower dose that is antimicrobial and antiseptic properties (0.25-1mgAgNPs/kg b.w.) through topical application but surprisingly it shows teratogenic effects like skeletal dysmorphology in 5, 10, 15, 20mg AgNPs/ kg b.w. dose and toxic consequences in 0.5 &1mg/kg b.w. dose through oral gavages as per studies by Benn et al. 14 and Sawosz et al. 15 reveals. This is definitely a big question for present research world who are really ignore about potential adverse effect. The question arise is why this potential adverse effect in higher dose but not in lower dose? Previously as per studies carried out by Stebunova et al. 16 & Kim et al. 17 revealed sub acute and long term exposure of nanosilver produces swelling of lung parenchyma or in-vitro toxicity there is no long term effect in chronic translocation of nanosilver into other organs.
Though lot of researches has been carried out on vertebrate animal to demonstrate nanosilver teratogenicity still there is lack of relevant data available till today to show teratogenic effect of repeated oral gavages of synthesized silver compound during prenatal to natal period which cause drastic teratogenic effect like infertility, intrauterine growth retardation and other anomalies among which skeletal dysmorphology is predominant.
McCauley et al. 18 did an experiment on pregnant cynomolgus monkeys in which he injected through vagina to uterus, 1% silver nitrate between 27 to 43days of pre natal period and surprisingly it cause vaginal bleeding in early stage of ingestion and he also observed termination of pregnancy but as an antagonistic effect of silver compound he also observed another victim of the particular experiment redelivered healthy and normal offspring after getting pregnant. As per studies by Blumberg & Carey 19 revealed & also observed a systemic aberration called argyria (Blue baby syndrome) due to chronic, part by part ingestion of total 6.4gm silver nitrate within 6month in past. Rosen man et al. 20 also did an experiment on old man worker by exposing silver dust on eye ball and respiratory tract & he observed irritation of the former and he also observed reduced capability of night vision in that worker. Moss et al. 21 also did the same experiment and found to some extent same complication. The period of exposure divided was 1-10years. Another experiment was carried out by Moss et al. 21 by exposing silver nitrate through inhalation in some worker in which he observed discoloration of the conjunctiva & cornea. William 22 also did the same and observed the same. As per recent studies by Chang et al. 23 revealed by giving oral gavages of AgNo3 to an old man dermal distress, endocrine disorder, hyperlipidaemia & hypertension with blue grey facial sign are observed. Tang & Xi 24 did an experiment on a very old man of about 80years by applying self medication of silver where they observed myoclonic seizures in that old victim both of them also found to some extent same complication.
Stepien et al. 25 & Korani et al. 26 study showed fatal consequences in higher dose Silver nitrate oral gavages administration along with liver, kidney and spleen toxicity histopathologically in medium dose administration, but still complete evidences are not available in plenty manner on teratogenicity of the silver compound on skeletal system. The present work was undertaken to study the effect of prenatal exposure of silver nano particle colloidal solution through repeated oral gavages on developing skeleton of mice embryos. We found delayed ossification of occipital bone which was also reported as per studies by Chernoff. 27 The skeletal dysmorphology observed were in the form of unossified temporal, frontal and occipital bones. Maxillary and mandibular hypoplasia, nasal bone hypoplasia, scoliosis, cleft , absence of segments of hip bone, failure of fusion of segments and large cases of increased intercostal spaces and other rib anomalies were also encountered during the course of the present study. Alizarin stain completely failed to reach up to tail region to stain the tail. There are plenty of data available among nano silver toxicity from various manufactured products, the present indicated dysmorphology of skeletal system of Swiss albino mice variety which is predominant among several types of teratogenicity executed till today. Studies by Stucky & Berry 28 revealed delayed skeletal growth our study also reported the same. Webster et al. 29 and Rasmussen & Christensen 30 also reported maxillary hypoplasia our study also reported the same. In our study maxillary and mandibular hypoplasia exhibited as either a flat mid face or a narrow snout that can be compared with the typical human FAS features i.e. a flat mid face associated with maxillary and mandibular hypoplasia. It can also be termed as a small burst and tear nose or a vanished or completely absent philtrum with a long gap between the thinned upper lip as per studies revealed by Clarren et al. 31 So keeping views and reporting of other scientists our study can be comparable with other study. In fact our study also showed different type of limb defects. As shown in Table 2 below.


Supercritical water synthesis of nano-particle catalyst on TiO2 and its application in supercritical water gasification of biomass

ABSTRACT Hydrogen production by catalytic gasification in supercritical water (SCW) is a promising way to utilise biomass resource. Supercritical water not only provides homogeneous and rapid reaction environment for the biomass gasification but also causes catalyst agglomeration problems. In order to prepare activity and stable catalyst for biomass gasification in supercritical water, supercritical water synthesis method was utilised and the preparation method was investigated. Ni, Co, Zn and Cu metal elements were loaded on TiO2 particles which was proved to be hythothermally steady in supercritical water. And nano-particles were successfully made. Based on gas chromatography/mass spectrometer (GC/MS), scanning electron microscopy, energy dispersive spectrometer (EDS) and X-ray diffraction analysis methods, it turned out that metal catalysts have a uniform spherical structure with diameter around 30 nm. Metal catalysts synthesised with supercritical hydrothermal method showed certain catalytic effects. Ni catalyst had the best performance in stability while Zn catalyst possessed highest hydrogen yield.

Introduction
There are abundant biofuels of different kinds all around the world. However, present utilisation of direct combustion causes severe environmental pollution, moreover, the efficiency of combustion is relatively low [1,2]. During combustion of biofuel, it is necessary to provide enough air because combustion needs a gaseous phase, therefore, great amount of inert N 2 lowers overall thermal efficiency. On the other hand, it dilutes concentration of pollutants, which causes expensive cost of centralised processing and collection. For fuels, which contain oxygen themselves, would significantly reduce the demanded quantity for air. As a result, it is easier to control pollution and emission, and improves thermal efficiency. Therefore, depolymerisation has great potential for the production of oxygenated fuels from biomass [3][4][5][6].
In the process of depolymerisation of biofuels, massive hydrogen is indispensable. It has great practical significance to produce hydrogen by gasification of depolymerisation wastes to make the whole conversion system independent. Among all the gasification methods, supercritical water (SCW) gasification takes the advantages of the unique physical and chemical properties of SCW, is suitable for the gasification of depolymerisation wastes because the high energy consuming drying process is not necessary [7][8][9][10][11].
The key for a more economical system relies on a proper catalyst with fine abilities in SCW [12,13]. Considering that normal catalysts are very easy to sinter and then lose their catalytic effects in SCW, right method to prepare catalyst is of great importance [14][15][16][17][18]. Hydrothermal synthesis in SCW has been a hot spot for SCW catalysts. Because since catalysts are made in the environment of SCW, catalysts have already been tested for stability during the synthesis [19][20][21][22][23]. Meanwhile, without any organic solvent evolved, it is definitely a green and sustainable chemical way. Because of unique physical and chemical properties of SCW, some oxides, which are hard to synthesise in normal ways, have a better way to synthesise. Not only what we get is single phase, but also it is possible to develop a uniform morphology and structure. In addition, through temperature and pressure adjustment, morphology can be directionally controlled [24][25][26][27].
In this paper, four metal elements, Zn, Ni, Cu and Co were loaded on TiO 2 particles by supercritical water synthesis. The influence of preparation temperature upon catalyst topography was investigated. Multiple analysis methods such as scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS) were conducted for the catalysts before and after reaction. Furfural was selected as the model compound of depolymerisation wastes. Based on gasification results of furfural in SCW, different metal catalysts were discussed and compared.

Apparatus and procedure
The synthesis and gasification processes were conducted in a high-throughput batch reactor system with six subsystems. The reactor was fabricated from Inconel 625 and was designed with a maximum temperature and pressure of 750 C and 30 MPa, respectively. The system contained a batch reactor, an electric furnace and a data acquisition station (DAS) of temperature and pressure. With a temperature controller, the electric furnace can operate at a desired temperature. The DAS can monitor and record the temperature inside the reactor using a thermocouple inserted inside the reactor [8,28,29].
First, metal salt solution was injected into the reactor with desired amount of TiO 2 particles. Calculated amount of deionised water was injected into the reactor with a syringe in order that desired supercritical water condition can be obtained. After that, a purging process was conducted to avoid influence of air or other gases. Next, the metal catalysts extracted from the reactor were dried in a small stove. Then, these metal catalysts were used in gasification of furfural in SCW for the evaluation of the catalysts.

Materials
Reagents are commercial products bought from special companies and their content information is listed in Table 1. Titanium sulphate was bought from Tianjin Guangfu Fine Chemical Research Institute with the content within 15.0%-20.0%. TiO 2 was used as catalyst carrier and was obtained from Northwest Institute for Non-ferrous Metal Research.

Characterisation and analytical methods
All samples were characterised by XRD using a PANalytical X'pert MPD Pro X automatic diffractometer, with the λ CuKa D 1.541836 A radiation. The volt and current is 40 kV and 40 mA, respectively. Powders were observed by SEM, which were conducted on a JEOL JSM-7800F electron microscope coupled with an energy dispersive spectroscope. The composition of the gaseous products was analysed by gas chromatography (Agilent 7890A) with thermal conductivity detectors. High-purity argon was used as the carrier gas.

Data interpretation
CE, GE, HE and YH 2 were used for the evaluation of the gasification results [10] in every catalytic situation.
CE D (total carbon in the gaseous products)/(total carbon in furfural) £ 100% GE D (mass of the gaseous products)/(mass of furfural) £ 100% HE D (total hydrogen in the gaseous products)/(total hydrogen in furfural) £ 100% YH 2 (Yield of H 2 ) D molar amount of H 2 after reaction/mass of furfural (mol/kg)

Results and discussion
TiO 2 was a common used catalyst carrier in supercritical water [30][31][32][33]. In order to synthesise TiO 2 -based catalyst particles in SCW, titanium sulphate was used as the precursor and the main pathway was showed as follows [34,35]: Since we choose TiO 2 as our carrier of metal catalyst, it is necessary to explore the stability under different temperature. To realise it, we put TiO 2 into reactor for 20 min under different temperatures: 400, 500, 600 and 700 C. Figure 1 shows the SEM images of TiO 2 particles under different temperature. It can be seen that in a temperature range of 400-600 C, TiO 2 particles remained spheroidal and nano-sized while they suddenly changed to have a massive laminar structure with less surface area at 700 C. It is possible to result in the devitalisation of the catalyst. Moreover, when it comes to economy, lower operating temperature often means lower energy for catalyst preparation. From Equation (1), sulphuric acid was formed after hydrolysis of Ti(SO 4 ) 2 . This kind of strong acid will lead to severe damage of reactor and thermal couple, especially at higher temperature. XRD and EDS analysis at 700 C also showed that there was element Fe on the surface of carrier. Therefore, acetic salts seemed to be a better option and acetic salts were used as the catalyst precursor in the following experiments.
The hydrothermal stability of TiO 2 was tested in supercritical water with the temperature of 400 C in autoclave for 20 min as seen in Figure 2. SEM images show a comparison of TiO 2 particles before and after exposure to supercritical water. Generally, there was almost no obvious difference in TiO 2 particle structure.
Acetic slats are our choice to be loaded on TiO 2 particles. The reaction pathway is shown as Equations (3)-(5). Through three steps, metal element can be dispersed on TiO 2 particle surface in the form of oxides. For quantification, mass of metal in acetic salt is 20 wt% of TiO 2 . Then we did XRD analysis to investigate the crystalline phase on TiO 2 particles as seen in Figure 3. Elements Zn and Ni existed in the form ZnO and NiO, respectively as expected. It is surprising to that Cu 2 O was generated instead of CuO. It can be explained by atomic orbit theory that 3d 10 structure is more stable in SCW. For the element Co, two kinds of cobalt titanate were produced.
Supercritical water gasification in autoclaves was conducted to evaluate the catalyst prepared. Furfural was selected as the model compound of the depolymerisation wastes. Mass of furfural and deionised water was 0.5 and 5 g, respectively. Mass of TiO 2 was 0.25 g and residence time was 20 min. After filtration and drying, residues are analysed by multiple characterisation methods. As we can see in Figure 4, the catalyst crystalline phase was ZnO and zinc titanate after reaction for the Zn situation. NiO remained stable through the SCW gasification. And Cu 2 O is all turned into elementary Cu. At the same time, two kinds of cobalt titanate become Co 2 TiO 4 only. Table 2 lists all changes for every element. In summary, except for Cu, it occurred to all the elements that chemical valence remained unchanged.
SEM and EDS analysis were used to investigate the microstructure of synthesised catalyst. Synthesised catalyst particles have an excellent dispersity under the scope of 100,000 times magnified view in Figure 5. The diameters of the catalysts were roughly 30 nm which was almost the same as the original TiO 2 .
Take Figure 5(d) for example, energy spectrum analysis is listed in Table 3. As can be seen in Table 3, elements Ti and O were observed to have the most amount on the surface. Meanwhile, element Co was successfully loaded on TiO 2 particles uniformly and supercritical water synthesis method was proved to be successful. Figure 6 shows SEM images of metal catalysts particles collected after furfural gasification in SCW, and the magnification was 5000 times. There appeared to be some bigger spherical particles on the surface of catalysts. EDS analysis was conducted to figure out that the content was mainly carbon as seen in Table 4. It could be that metal catalyst accelerates the precursor of coke during the gasification of furfural in SCW. This kind of precursor probably grows up to coke particles centred on metal  catalyst particles. According to SEM image, diameter of coke particles is around 1 micrometre. Figure 7 shows the gasification results of furfural with and without different metal elements catalysts. The fraction of H 2 in gaseous products was only 6.34% and there appeared to be no methane without catalyst. In comparison, each catalyst improved HE vastly and some metal catalysts showed unique effect. At the same time, all catalysts

Conclusions
Nano metal catalysts based on TiO 2 particles were successfully prepared by supercritical water synthesis. 400 C was approved to be the optimal preparation temperature because it can promote the generation of catalyst and reduce energy consumption. The stability of catalysts was tested over 20 min in supercritical water and Zn, Ni, Cu, and Co were proved to be dispersed on the surface of TiO 2 particles. According to XRD analysis, Ni performed the best stability without crystalline phase change. Although there happened to be some coke covered on the catalyst particles, gasification results of furfural in SCW were still much better than those without catalyst. By comparison, zinc catalyst showed best catalytic effects among all catalysts. Single metal catalysts carried on TiO2 can be successful prepared by supercritical water synthesis and performed catalytic effect for furfural gasification in supercritical water.

Disclosure statement
No potential conflict of interest was reported by the authors.


Fischer – Tropsch synthesis: e ﬀ ect of ammonia on product selectivities for a Pt promoted Co/alumina catalyst

The e ﬀ ects of co-fed ammonia in synthesis gas on the activity and product selectivities of a typical cobalt catalyst (0.5% Pt – 25% Co/Al 2 O 3 ) were investigated during the Fischer – Tropsch synthesis using a continuously stirred tank reactor (CSTR). The product selectivities were compared at a similar CO conversion level for various concentrations (10 – 1000 ppmv) of ammonia, as well as clean (un-poisoned) conditions. The addition of 10 – 1000 ppmv ammonia (concentration of ammonia with respect to the syngas feed) signi ﬁ cantly decreased activity; the percentage of deactivation was similar ( (cid:1) 40%) for the various concentrations of ammonia used. At similar CO conversions, the addition of ammonia caused an increase in ole ﬁ n selectivity and the corresponding para ﬃ n and alcohol selectivities were decreased compared to the ammonia free synthesis conditions. Ole ﬁ n selectivity increased with increasing concentration of ammonia, and the para ﬃ n and alcohol selectivities were decreased with increasing ammonia concentration. At similar CO conversions, ammonia addition exhibited a positive e ﬀ ect on hydrocarbon selectivity ( i.e. , lower light gas products and higher C 5 +) and also light gas product selectivities (C 1 – C 4 ) were decreased and C 5 + selectivity increased with increasing concentration of ammonia compared to ammonia free conditions.

Introduction
Fischer-Tropsch Synthesis (FTS) is a promising route for the transformation of synthesis gas (CO + H 2 , or syngas) into valuable hydrocarbons (gasoline, diesel, chemicals, etc.). It is suitable for the production of synthetic fuels from both fossil (e.g., coal, natural gas) and renewable (e.g., biomass) resources. [1][2][3] This reaction produces a wide variety of products, from gases to heavy waxes. FTS is catalyzed by several transition metals including Fe, Co, and Ru, whereas only Co and Fe are commonly employed as reasonable commercial catalysts for the process. However, cobalt catalysts may feature high stability and activity, along with a lower deactivation rate, compared to iron catalysts, 4 but require a narrower range of operating temperatures and pressures, because any overheat shis the reaction towards methane formation and decreases the desired liquid product selectivity. 5,6 A gasication process is used to convert coal or biomass feedstocks into synthesis gas (CO and H 2 mixture), which undergoes the Fischer-Tropsch reaction aer H 2 /CO ratio adjustment and CO 2 removal. However synthesis gas also contains various impurities that must be removed in order to prevent FT catalyst poisoning. Biomass-derived synthesis gas can contain both organic and inorganic impurities such as tars, benzene, toluene, xylene, NH 3 , HCN, H 2 S, COS, HCl, volatile metals, dust, and soot. 7 Coal, which originates from biomass, contains many of the same inorganic impurities as found in biomass. 8 Due to the variety of feedstocks that can be processed, signicant variations in the composition of synthesis gas can be expected. This affects the nature of the impurities that are present (element, speciation), as well as their relative contents. Moreover, due to the high sensitivity of FT catalysts, severe specications regarding syngas purity are required. Iron and cobalt catalysts share the sensitivity toward some, but not all, of the impurities commonly found in coal and biomass-derived syngas.
Among these impurities, high concentrations of sulfur compounds ($500 ppbv) may cause irreversible deactivation of cobalt and iron-based catalysts during FT synthesis. [9][10][11][12] The effect of ammonia on FTS has been reported in a few studies over iron and cobalt catalysts and the reported results, however, have been rather contradictory. [13][14][15][16][17][18] Claeys et al. 13 reported that co-feeding of up to 25% NH 3 in syngas did not affect FT activity; similarly Borg et al. 14 reported that 4.2 ppmv of ammonia does not have an impact for a CoRe/Al 2 O 3 catalyst. An Exxon patent 15 claims that a combined concentration of 100 ppb of NH 3 and HCN in syngas will result in a catalyst half-life of only 4 days for a supported cobalt catalyst in a slurry reactor. However, the patent also indicates that hydrogen treatment of the catalyst can restore the initial activity. Van Berge 16 reported that cobalt catalysts were rapidly but reversibly deactivated by HCN and NH 3 . In a recent investigation, Rausch et al. 17 indicated that ammonia had a signicant effect on the cobalt catalyst. Ordomsky et al. 18 reported that the addition of NH 3 and CH 3 CN during FT synthesis has a signicant irreversible deactivation on supported cobalt catalysts, whereas no noticeable deactivation was observed with supported iron catalysts.
In our previous investigations, [19][20][21][22] we reported that an iron catalyst was quite resistant to high levels (200 ppmw) of ammonia, 19 whereas a cobalt catalyst underwent signicant deactivation with parts per million levels of ammonia, [20][21][22] regardless of whether ammonia gas or ammonium hydroxide was used. The addition of ammonia caused signicant deactivation for alumina, titania and silica supported cobalt catalysts, but the rate of deactivation was higher for the silica-supported catalyst compared to the alumina and titania supported catalysts. 20 In most studies reported in the open literature, the effect of ammonia on product selectivities over cobalt catalysts has not been reported at similar CO conversion levels. The aim of the present study is to compare the product selectivities at similar CO conversion levels for various concentrations of cofed ammonia (10-1000 ppmv) as well as under clean conditions for a Pt-promoted Co/alumina catalyst.

Catalyst preparation
Sasol/Catalox alumina (high purity g-alumina, 140 m 2 g À1 ) was used as the support for the cobalt catalyst. The catalyst was prepared by a slurry impregnation method, and cobalt nitrate was the precursor. In this method, which follows a Sasol patent, 23 the ratio of the volume of solution used to the weight of alumina was 1 : 1, such that the volume of solution was approximately 2.5 times the pore volume of the catalyst. Two impregnation steps were used, each to load 12.5% of Co by weight. Aer the second impregnation/drying step, the catalyst was calcined under air ow at 350 C. The promoter was added by incipient wetness impregnation (IWI), and the precursor utilized for noble metal addition was tetraammineplatinum(II) nitrate. Aer Pt addition, the sample was dried and calcined again using the same conditions as used previously.

Catalyst characterization
2.2.1 BET surface area and pore size measurements. To characterize the ammonia-exposed catalysts, the end-of-run catalyst along with wax was transferred to an air-free environment (inert gas lled chamber). The catalyst sample was diluted with hot ortho-xylene to remove the high molecular weight FTwax products. It was not possible to completely remove the FT-wax from the catalyst particles by this method. However, the remaining wax acts as a protective barrier for the air-sensitive catalyst particles. The extracted catalyst was treated with 1% O 2 /N 2 at 300 C for 4 h to remove the wax product formed from FTS.
BET surface area and porosity measurements of the calcined and ammonia exposed catalysts were conducted using a Micromeritics 3-Flex unit. Before performing the test, the temperature was gradually ramped to 160 C and the sample was evacuated for at least 12 h to approximately 50 mTorr.
2.2.2 Hydrogen chemisorption and pulse reoxidation. Hydrogen chemisorption was conducted using temperature programmed desorption (TPD), also measured with the Zeton Altamira AMI-200 instrument. The sample weight was typically $0.22 g. The catalyst was activated in a ow of 10 cm 3 min À1 of H 2 mixed with 20 cm 3 min À1 of argon at 350 C for 10 h, and then cooled under owing Ar to 85 C. A gas mixture of 10 cm 3 min À1 H 2 and 20 cm 3 min À1 Ar was owed at 85 C for 15 min to chemisorb hydrogen to the catalyst surface. The sample was then held at 85 C under 30 cm 3 min À1 owing of Ar for 30 min to remove and/or prevent adsorption of weakly bound species prior to increasing the temperature slowly to 350 C, the reduction temperature of the catalyst. The catalyst was held under owing argon to desorb any remaining chemisorbed hydrogen until the TCD signal returned to baseline. Further details of the procedure are provided elsewhere. 24 For the ammonia exposed catalyst, the catalyst was initially activated in a ow of 10 cm 3 min À1 of H 2 mixed with 20 cm 3 min À1 of argon at 350 C for 10 h, and then cooled to 220 C under owing Ar, aer which ammonia gas (1% NH 3 balanced nitrogen, 50 cm 3 min À1 ) was owed for an hour. The sample was then cooled to 85 C under owing of Ar. A gas mixture of 10 cm 3 min À1 H 2 and 20 cm 3 min À1 Ar was owed at 85 C for 15 min to chemisorb hydrogen to the catalyst surface. The sample was then held at 85 C under owing Ar to remove and/ or prevent adsorption of weakly bound species prior to increasing the temperature slowly to 350 C, the reduction temperature of the catalyst.

Catalytic activity testing
FTS reaction experiments were conducted using a 1 L continuously stirred tank reactor (CSTR) equipped with a magnetically driven stirrer with turbine impeller, a gas-inlet line, and a vapor outlet line with a stainless steel (SS) fritted lter (2 mm) placed external to the reactor. A tube tted with a SS fritted lter (0.5 mm opening) extending below the liquid level of the reactor was used to withdraw reactor wax (i.e., rewax, which is solid at room temperature), thereby maintaining a relatively constant liquid level in the reactor. Separate mass ow controllers were used to control the ow rates of hydrogen and carbon monoxide. Carbon monoxide, prior to use, was passed through a vessel containing lead oxide on alumina to remove traces of iron carbonyls. The gases were premixed in an equalization vessel and fed to the CSTR below the stirrer, which was operated at 750 rpm. The reactor temperature was maintained constant (AE1 C) using a temperature controller.
The catalyst ($12.0 g) was ground and sieved to 63-106 mm before loading into a xed-bed reactor for 12 h of ex situ reduction at 350 C and atmospheric pressure using a gas mixture of H 2 /He (60 NL per h) with a molar ratio of 1 : 3. The reduced catalyst ($10.0 g) was transferred to a 1 L CSTR containing 310 g of melted Polywax 3000 under the protection of inert nitrogen gas. The reactor used for the reduction was weighed before and aer reduction and aer catalyst transfer in order to obtain an accurate amount of catalyst that was added. The transferred catalyst was further reduced in situ at 230 C at atmospheric pressure using pure hydrogen (20 NL per h) for another 24 h before starting the FT reaction. In this study, the FTS conditions used were 220 C, 1.99 MPa, H 2 /CO ¼ 2, and a stirrer speed of 750 rpm.
Gas, water, oil, light wax, and heavy wax samples were collected daily and analyzed. Heavy wax samples were collected in a 200 C hot trap connected to the lter containing dip tube. The vapor phase in the region above the reactor slurry passed continuously to the warm (100 C) and then the cold (0 C) traps located external to the reactor. The light wax and water mixture were collected daily from the warm trap and an oil plus water sample from the cold trap. Tail-gas from the cold trap was analyzed with an online HP Quad Series Micro GC, providing molar compositions of C 1 -C 5 olens and paraffins, as well as H 2 , CO and CO 2 . The analysis of the aqueous phase used an SRI 8610C GC with a thermal conductivity detector. The organic products were analyzed with an Agilent 6890 GC with a ame ionization detector and a 60 m DB-5 column. Hydrogen and carbon monoxide conversions were calculated based on GC analysis of the gas products, the gas feed rate, and the gas ow that was measured at the outlet of the reactor.

Results and discussion
To maintain experimental control, similar activation and reaction conditions (partial pressures of CO and H 2 ) were used at various concentrations of ammonia exposure for a Pt-Co/ alumina catalyst. Aer attaining steady state CO conversion, ammonia gas (10 to 1000 ppmv) was added to the syngas feed. The addition of ammonia (10-1000 ppmv) caused a signicant initial drop in CO conversion and then the CO conversion remained relatively constant with addition of ammonia following the initial drop. More importantly, CO conversion did not recover to the expected value, even aer termination of ammonia addition under normal FTS conditions, indicating that the catalyst had deactivated (i.e., NH x species are strongly adsorbed to deactivate cobalt active sites). 20 The relative CO conversions for a Pt-promoted Co/alumina catalyst at various concentrations of ammonia compared to the pre-exposure conditions are shown in Fig. 1. This gure shows that the extent of deactivation is similar (AE3%) for the various concentrations of ammonia used (10-1000 ppmv). The similar extents of poisoning are consistent with the chemisorption of the same amount of ammonia at the pressure represented by 10-1000 ppmv of ammonia.
The effect of ammonia on product selectivities over a Ptpromoted Co/alumina catalyst at various concentrations of ammonia is shown in Table 1. In FT synthesis, it is well known that the conversion level usually inuences the selectivity, in part due to increasing partial pressure of water and by increasing the partial pressure of reactants. 25,26 It is therefore important to compare the catalysts at a similar CO conversion level.
Experiments were conducted for the un-poisoned and ammonia exposed catalyst at various concentrations. The addition of 10 ppmv ammonia caused an increase in olen selectivity from 16.7 to 21.3%, while paraffin selectivity slightly decreased to 77.5% from 78.3% and alcohol selectivity decreased to 1.2% from 5.0%. For 100 ppmv addition of ammonia, olen selectivity increased to 23.3% from 16.7%, paraffin selectivity decreased to 74.7 from 78.3% and alcohol selectivity decreased to 2.0% from 5.0%. Similarly, the addition of 1000 ppmv ammonia caused an increase in olen selectivity to 26.8% from 16.7%, while paraffin selectivity decreased to 72.7% from 80.4% and alcohol selectivity decreased to 0.5% from 2.7%. At similar CO conversions, the addition of ammonia caused an increase in olen selectivity and corresponding decreases in alcohol and paraffin selectivities compared to the ammonia free synthesis condition. With increasing concentration of ammonia, the extents were increased with similar trends (i.e., increase in olen selectivity, decreases in paraffin and alcohol selectivities) compared to the ammonia free synthesis condition.
The effect of ammonia on hydrocarbon selectivities at similar CO conversions were compared for the unpoisoned and  ammonia poisoned catalysts, which are shown in Table 2. The addition of 10 ppmv ammonia decreased the methane selectivity to 7.6% from 8.7% and the corresponding C 5 + selectivity increased to 83.9% from 81.5%. For 100 ppmv addition of ammonia conditions, methane selectivity dropped to 6.7% from 8.7% and the C 5 + selectivity was enhanced to 85.9% from 81.5%. Similarly, the addition of 1000 ppmv ammonia caused a signicant decrease in methane selectivity (6.5% from 9.3%) and the corresponding C 5 + selectivity increased to 86.2% from 80.2%. These results clearly indicate that addition of ammonia signicantly decreases the lower hydrocarbons selectivity (C 1 -C 4 ) and increases the corresponding higher hydrocarbon (C 5 +) selectivity. With increasing ammonia concentrations (10 to 1000 ppmv), lower hydrocarbon selectivities decreased and higher hydrocarbons selectivity increased. A loading of 0.5% Pt is typical of 25% Co/Al 2 O 3 research catalysts for facilitating the reduction of cobalt oxides that strongly interact with the support. 24,27 EXAFS experiments have revealed that Pt is in contact with Co at the atomic level, with no Pt-Pt coordination observed; 28 moreover, comparing selectivity at constant conversion, Pt did not signicantly alter the selectivity of FTS compared to an un-promoted catalyst. 29 That is, the role of Pt is primarily to increase the extent of cobalt reduction to, in turn, increase the surface Co metal active site density. In our earlier study, 22 the effect of ammonia over Pt promoted and un-promoted 20% Co/SiO 2 catalysts was investigated. Similar trends were observed for the un-promoted and Pt-promoted silica supported cobalt catalysts, and the Pt promoted catalyst deactivated more than the un-promoted catalyst during the ammonia exposure. Regarding the conversion and selectivity trends, Pt promoted Co/SiO 2 did not display any additional benets or drawbacks relative to the un-promoted silica supported cobalt catalyst.
The 1-olen contents in total hydrocarbons as a function of carbon number at similar CO conversions for the un-poisoned and 1000 ppmv ammonia poisoned catalysts are shown in Fig. 2. At similar CO conversion level, the 1-olen content (selectivity) of the ammonia exposed catalyst is higher than for the unpoisoned catalyst at all carbon numbers (C 2 -C 17 ). Ethylene is more reactive than other low molecular weight 1-olens, and thus its selectivity is correspondingly lower. The low ratio of ethylene to ethane was probably due to the rapid readsorption 30 and hydrogenation and/or incorporation of ethylene for chain propagation. 31 Linear 1-olens are known to be the main primary organic products of FTS. Once formed, they can readsorb from the catalyst surface and undergo secondary reactions: hydrogenation, isomerization, reinsertion, hydrogenolysis and hydroformylation. The observed product can be impacted by secondary reactions. Olen secondary reactions strongly depend on reaction conditions (temperature, partial pressures of H 2 , CO and water and residence time), the catalyst used and the olen chain length. The olen content (from C 3 onwards) decreased with increasing carbon number for both unpoisoned and ammonia poisoned catalysts. The decrease in olen content with increasing carbon number (for C 3 + hydrocarbons) has been attributed to differences in reactivity, intraparticle diffusional effects, residence time differences due to vapor pressure differences and solubility variation with molecular weight. 6,32-36 Diffusivity decreases with increases in molecular weights of the species, and higher molecular weight 1-olens have longer residence times within catalyst pores, thus increasing the probability for secondary reactions. [32][33][34] Increases in solubility with increases in carbon number also result in increased residence times for higher molecular weight hydrocarbons in the reactor as well as their higher liquid phase concentrations, resulting in a greater extent of secondary reactions. 6,34-36 Due to diffusional restrictions, larger a-olens spend longer times in a pore, which increases the probability of their readsorption on FTS active sites before exiting the pore. In a typical liquid product produced from cobalt catalyst, olens will appear around up to carbon number 25, whereas paraffins will appear above about carbon number 35. Olens are not observed above carbon number $25 in the liquid products because their long pore residence time ensures that olens will readsorb many times; only chains that terminate as unreactive paraffins exit the catalyst. Thus the higher paraffinic content of larger hydrocarbons is not the result of direct hydrogenation of a-olens to the corresponding paraffin of equal size, 32,33 but reects instead the enhanced readsorption of a-olens and surface chain initiation steps that lead to their ultimate desorption as paraffins.  The decrease in methane selectivity along with higher 1-olen content of the ammonia exposed catalysts might be due to the suppression of secondary reactions of olens (such as hydrogenation, isomerization, re-insertion and hydroformylation). Two additional experiments were carried out to further conrm the suppression of secondary hydrogenation reaction of olens, by co-feeding 1-decene to the unpoisoned and ammonia poisoned catalysts during FT synthesis. Gas chromatographic results and 1-decene conversion of the unpoisoned and 1000 ppmv ammonia exposed conditions are shown in Fig. 3. The conversion of 1-decene was calculated from its rate of consumption assuming that the FT product formation rate was not affected by 1-decene addition. The added decene is primarily converted to the corresponding linear paraffin (decane). The activity and selectivity results of 1-decene under FT conditions for the unpoisoned and ammonia exposed catalysts are shown in Table 3. The 1-decene conversion of the ammonia exposed (1000 ppmv) catalyst was found to be 46.0%. The selectivity of the hydrogenation product (decane) was found to be 90.0% and the remaining 10.0% selectivity was to isomerization products (cis and trans isomers of 2-decene). For the clean catalyst, 85.4% 1-decene conversion was observed, and the selectivities of the hydrogenation and isomerization products were found to be 91.0% and 9.0%, respectively. These results clearly reveal that addition of ammonia caused a significant suppression of secondary reactions of olens such as hydrogenation and isomerization for the supported cobalt catalysts. The 1-decene addition during FT over unpoisoned and  ammonia exposed catalysts does not have any signicant impact on methane selectivity. This suggests that hydrogenolysis leading to methane from added 1-decene and the olens formed during synthesis played a negligible role. The effect of ammonia on FTS activity and selectivity of cobalt catalysts is unclear; some researchers have reported no effect, 13,14 while others have reported that ammonia had a signicant impact. [15][16][17][18]20,21 In the open literature, most of the studies have not reported the product selectivities at similar CO conversion level. Recently, Rausch et al. 17 reported that ammonia addition had a signicant impact on activity and product selectivity as well. The activity of the catalyst was decreased with increasing the concentration of ammonia. The addition of ammonia resulted in an increase in olen selectivity and decreases in paraffin and alcohol selectivities. Ordomsky et al. 18 also reported that small amounts of acetonitrile and ammonia affected the FTS activity and selectivity of the cobalt catalysts. The deactivation of the cobalt catalyst was explained by possible formation of cobalt nitride, which may be responsible for the selective blocking of methanation sites. Lower methane and higher C 5 + selectivity of the cobalt catalysts was explained by a decrease in intrinsic hydrogenation activity of cobalt sites aer the formation of cobalt nitride. The effect of ammonia on product selectivity of Pt-Co/alumina catalyst is similar to the effect of potassium promoter to an iron-based FT catalysts. As we mentioned in our earlier studies, 37,38 the addition of potassium to the iron catalysts increases the strength of CO adsorption and suppresses H 2 . This results in a high CO/H concentration ratio on the catalyst surface and consequently leads to a lower hydrogenation activity. A lower hydrogenation activity would suppress the hydrogenation of surface CH x species and also secondary hydrogenation of olens. As a result, low selectivity to methane and high selectivity to higher hydrocarbons and olens was observed.
To understand the reason for changes in activity and product selectivities following ammonia exposure, the catalysts were investigated by characterizing the freshly calcined and ammonia exposed catalysts by using BET surface area and hydrogen chemisorption techniques. BET surface area and pore size distribution results of the Pt-Co/alumina catalysts are presented in Table 4. The BET surface area and pore size distribution results of the freshly calcined, FT used, ammonia exposed during FT and the regenerated (aer ammonia exposure mild in situ H 2 treated) catalysts had similar values (AE3%, within experimental error range). These values suggest that ammonia exposure during FTS does not cause any signicant structural changes of the catalyst (e.g., pore collapse) as was the case with Co/SiO 2 catalysts. 20 The hydrogen chemisorption results of the freshly calcined catalyst are shown in Table 5. Ammonia addition may decrease the reactivity of hydrogen. For a hydrogen activated freshly calcined clean catalyst the H 2 -TPD up to 325 C shows complete desorption of hydrogen (139 mmol g À1 ), while for the ammonia pretreated catalyst, only 88% of the H 2 had desorbed (122 mmol g À1 ) by that temperature (325 C). Both fresh and ammonia-exposed catalysts desorbed relatively similar amounts of hydrogen aer a 30 min hold at 350 C. During FT reaction, continuous addition of ammonia caused a 40% drop in activity, but in TPD, hydrogen desorbed was lowered by only 12%. Note that we owed H 2 at 85 C for 15 min during TPD measurement, which may have removed a fraction of the NH x species from the surface prior to the TPD, so the result may not reect the effect of constant NH 3 addition during FT.
The effect of ammonia on the catalytic activity can be explained by the adsorption of NH x species on the cobalt surface which may or may not form surface cobalt nitride and which severely decreases the ability to adsorb the reactants (hydrogen and CO). This changes the selectivity due to a coverage of NH x species on metal sites that normally adsorb hydrogen, creating a relatively hydrogen poor surface. Ordomsky et al. 18 proposed that the formation of cobalt nitride might be responsible for the changes in activity and selectivity following ammonia exposure. There are two likely scenarios for  the benecial impacts on selectivity (lower methane and higher C 5 +) with ammonia addition for the alumina supported cobalt catalyst; one is that ammonia may inhibit the methanation sites responsible for the deviation of methane selectivity above that produced from ASF kinetics; another possibility is that ammonia inhibits termination of chain growth. The evidence for the former is a slight decrease in the deviation from the ASF distribution for methane and the evidence for the latter is an increase in the value of a. The formation of surface cobalt nitride or adsorbed NH x might block the methanation sites, which results in lower methane and higher C 5 + selectivity. In earlier studies 39,40 it was reported that cobalt nitride forms in the presence of ammonia over alumina and silica supported cobalt catalysts. In one of our very recent investigations, 41 we studied the effect of COS on the activity and selectivity of a Pt-Co/alumina catalyst. The addition of COS caused a signicant irreversible deactivation, and lower hydrocarbon (C 1 -C 4 ) selectivities increased and higher hydrocarbon (C 5 +) selectivity decreased compared with unpoisoned catalyst at similar CO conversion level. S K-edge XANES analysis of the COS poisoned cobalt catalyst indicated that the formation of cobalt sulde (Co-S) was responsible for the decrease in CO conversion and change in product selectivities. Similarly, in the present work, the addition of ammonia over cobalt catalyst might cause the formation of cobalt nitride, which might be responsible for the changes in activity and selectivity. In our previous investigations, 20,21 we reported that the addition of ammonia caused a signicant decrease in CO conversion over supported cobalt catalysts and that catalyst activity did not recover even aer termination of ammonia addition under normal FT conditions. However, the results for this study show that the activity of the Pt-Co/alumina catalyst virtually recovered to the initial values aer the catalyst had been subjected to a mild in situ hydrogen treatment (i.e., similar FTS conditions but the carbon monoxide was switched off) for a 24 h period (Fig. 4). The addition of ammonia caused a signicant decrease in activity and also altered the product selectivity. Aer termination of ammonia addition, the activity and selectivities did not recover to the initial values but remained at the poisoned level; this may be explained by blocking of active sites by chemisorbed surface NH x species, thereby restricting the adsorption of reactants (H 2 and CO). These surface NH x species are not removed aer the termination of the ammonia feeding. However, mild in situ hydrogen treatment removed the surface NH x species and the activity and product selectivity recovered to the initial values. The olen/ paraffin ratio of the Pt-Co/alumina prior to, during and aer addition of ammonia and aer hydrogen treatment conditions are shown in Fig. 5. As we discussed earlier, the addition of ammonia resulted in an increase in the olen/paraffin ratio or olen content relative to ammonia free synthesis conditions. Aer the termination of ammonia addition, the catalyst did not recover the activity and product selectivities (hydrocarbons, and olen/paraffin ratio) as well. However, aer a mild in situ hydrogen treatment, the activity and product selectivities recovered almost to their initial values ( Fig. 4 and 5, respectively). These results indicate that the effect of ammonia is able to be reversed (activity and product selectivity) with mild in situ hydrogen treatment over a Pt-Co/alumina catalyst.

Conclusions
The effects of ammonia on activity and product selectivities were investigated by varying the concentration of ammonia during Fischer-Tropsch synthesis. The addition of ammonia caused a signicant step drop in CO conversion, and the extent of decrease in CO conversion was similar for all concentrations (10 to 1000 ppmv) tested in this study. The product selectivities were compared at similar CO conversion level for unpoisoned and ammonia poisoned catalysts. At similar CO conversion, the addition of ammonia caused an increase in olen selectivity and decreased the corresponding paraffin and alcohol selectivities. With increasing concentration of ammonia, the formation of olens increased and the formation of paraffins  and alcohols decreased. The hydrocarbon selectivity was also signicantly impacted by the addition of ammonia; lower hydrocarbon selectivities (C 1 -C 4 ) decreased and the corresponding higher hydrocarbon selectivity (C 5 +) increased for the ammonia exposed catalysts compared to under clean conditions at similar CO conversions. The decrease in methane selectivity accompanied by the higher olen content of the ammonia poisoned catalyst is due to the suppression of secondary reactions of olens (i.e., hydrogenation and isomerization). The suppression of secondary reactions with ammonia addition might be due to the formation of cobalt nitride. The effects of ammonia on activity and product selectivities were able to be reversed (virtually recovered to the initial values) with mild in situ hydrogen treatment over an ammonia exposed Pt-Co/alumina catalyst.