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Fullerenes Synthesis Extraction and Purification

There are many methods to synthesis C60 and C70 in gram quantities in the laboratory. In addition, higher mass fullerenes (larger fullerenes molecules) can be produced and isolated , albeit in very small amounts [1].
Most methods for generation of large quantities of fullerenes produce a mixture of impurity molecules and stable fullerenes. For this reason, fullerene synthesis must be followed by processes of extraction and purification of fullerenes from impurities according to mass [1].
Synthesis of Fullerenes: Fullerene molecules can be synthesized in the laboratory in a wide variety of methods, all involving the formation of a carbon- rich vapour [1].
Early methods used laser evaporation technique which produces very small quantities of fullerenes [1]. The later approaches involve an electric discharge between graphite electrodes in He gas [10].
Laser Evaporation Technique:
This method was used in 1984 for the first time by Rohlfing and others [8]. They noticed that carbon cluster Cn with a huge number of carbon atoms (more than 190) could be produced [9]. In 1985 Kroto, Smalley and co- workers used this technique to generate and detect the most stable carbon clusters [7].
This method involves vaporization of carbon species from the rotating graphite disk into a high density helium, using a Nd:YAG laser operation at 532nm, (fig2). The resulting carbon clusters were analysised by time -of- flight mass spectrometry. The first observation of the mass of C60 was a 720 amu peak. Although this approach produces minute quantities of fullerenes, it is still essential if when we use later modification. This modification will help to heat the dusk of graphite. Therefore, it gives remarkable control of fullerene distribution and the generation of specific fullerenes [2].
Arc Evaporation:
There is no doubt that this technique is an efficient way to produce gram quantities of fullerenes in the laboratory [1]. For the formation of fullerenes by this technique, an arc is struck between two graphite electrodes in atmosphere of 100~200 torr of He. The contact between the electrodes is maintained by the influence of gravity. The apparatus is surrounded by water to cool the soot to achieve the resulting soot which may contain approximately 10-15% of soluble fullerenes [2].
The first design by Wudl and co- workers used a pyrex cylinder for the vacuum shroud. Although this gives a suitable method for visual inspection of the graphite electrodes through the well, the glass cylinder is easily damaged. For this reason, it is appropriate to change it with a stainsteel cylinder with a window [1].
Fullerene Extraction:
In this process of fullerenes production, soluble impurity molecules and insoluble nanoscale carbon soot are generated with soluble fullerenes. Two effective methods are used to extract the fullerenes from the soot [1].
Solvent Methods:
Solvent method is the most common method is used to dissolve the fullerenes in benzene, toluene (preferred over benzene due to its toxicity is lower) or other suitable solvent. However, the solvent also contains other soluble hydrocarbon impurities [2]. It can be separated soot and other insoluble molecules from the solution by filtration. The early method used Soxhelt extraction in a hot solvent to remove fullerenes from the soot. This technique is used where the molecules to be extracted from the solid state are soluble in organic solvent, such as polyaromatic hydrocarbons (PAHs) from coal. This apparatus consists of double thimble containing soot, fullerenes and other materials and at the bottom the solvent is boiled in the flask. The solvent vapors and rises to condense in the condenser unit, the solvent distills then the solution passes through the thimble wall. The solution which contains the extracted molecules returns to the flask. The molecules that are not soluble in the solvent remain in the thimble. Another alternative method, the soot is separated in tetrahydrofuran (THF) at room temperature before sonicating the soot in an ultrasonic bath for 20 minute. Removing insoluble molecules by filtration and a rotary evaporator at 50°C are used to remove THF from the fullerenes. It can be noticed that the higher boiling point solvent and more polar isolate the higher mass fullerenes [1].
Sublimation Methods:
It can be sublimated microcrystalline C60 and C70 powder at low temperature Ts~350°C (C60) and Ts~460°C (C70). For this reason, C60 and C70 can be separated directly from the soot without introducing solvents, such as benzene, toluene, carbon disulfide or hexane. This method provides a beneficial alternative to solvent extraction for some cases which are sensitive to contamination of solvent in the sample. In this approach, the raw soot is placed in a quartz tube and the whole apparatus is heated in a furnace. Dynamic pumping is preferred because it is likely the soot may contain polyaromatic hydrocarbons impurities. The raw arc soot in the end of tube is kept at the highest temperature T~600-700° C. The higher mass fullerenes sublimate from the soot which then condenses in the colder section of the tube. Since the sublimation temperature of C70 and higher fullerenes are higher than that of C60, they will condense closer to the soot. The production of a C60 molecular beam from a microcrystalline mixture of C60 and C70 depends on the difference in sublimation temperature between C60 and C70. This microcrystalline mixture is placed in a dynamic vacuum and is heated above the sublimation temperature of C60. The sublimation rate for C60 in vacuum at T~400 °C is favored by a factor of 20 over that C70. A pure molecular beam of C60 can be obtained, because C70 is a factor of ~ 7 less abundant in arc soot than C60 [1].
Kratschmer et al [11] used the method of directly subliming fullerenes from the solid material. However, this does not provide pure fullerenes.
Fullerene Purification:
The previous methods of extraction may bring impurity molecules with the most stable fullerenes. The step of chemical purification must be carried out, if a pure fullerene microcrystalline powder or solution is desired. The step involves sublimation methods based on temperature gradients and solvent methods based on liquid chromatography. Fullerene purification means the separation of the different fullerenes in the fullerene extract into C60, C70, C76, C84 etc. Sensitive tools, such as liquid chromatography, mass spectrometry, nuclear magnetic resonance (NMR), optical absorption spectroscopy and infrared [1].
Solvent Methods:
The main technique for fullerene purification is liquid chromatography (LC). LC is a wet chemistry method which includes a solution ( called the mobile phase ) of a molecular mixture. This solution is forced to pass through a column filled with a high surface area solid (called the stationary phase ). The separation of fractions is verified qualitatively by the comparison of the observed optical spectra, vibration spectra and NMR data or by color ( magenta or purple for C60 in toluene and reddish- orange for C70 in toluene). Liquid chromatography separates molecules according to their weights. Moreover, this technique can be utilized to separate a single allotrope, such as C76, or to isolate isomers with different molecular shapes but having the same molecular weight, such as separating C78 with C2? symmetry from C78 with D3 symmetry [1].
The liquid chromatography process involves chemical or physical interactions between a particular molecule and the stationary phase. This interaction reduces (or raises) the rate of migration for that molecule through the column or raises (or reduces) the retention time for that molecule.
Remarkable chemical or physical differences for the molecular species, such as surface absorption, shape and mass are important to provide a clear chromatographic separation. Early approaches to C60, C70, and higher fullerenes purification included flash column chromatography of the raw fullerene in a column packed with neutral alumina as the stationary phase and hexane/toluene ( 95/5 volume % ) as the mobile phase. Although this process was found useful, it used abundant quantities of solvent that was difficult to recycle [1].
One of the first important development to this method was high performance liquid chromatography (HPLC).

Sewage Treatment Plant Power Generation

Answer 1: Sewage treatment plants could be a power house of the future. Sewage contains a number of diverse chemical compounds which can be with the help of microbes converted to useful commodities. The proposed sewage plant is as under.

The waste water sludge of the plant is where a microbiologist is interested in to utilize the components of the sludge and modify them microbiologically. For the correct type of fermentation we first of all need the microbes which operate in the same environment and produce the desired products. For this the sampling of the microbes from the sludge is the first step. After sampling they will be isolated with the help of biochemical tests based on the characteristic property we want to utilize. Here in this case we can grow;
Methanogens
Hydrogen gas producers
Organic compound synthesizers
Heavy metal detoxifiers
Safe effluent water
Sludge as manure
Microbial Pathways:
Methanogenesis:

The microbes usually found for methane gas production are Methanosarcina, they have the enzyme machinery suitable for the methane gas production.
Hydrogen Gas Producers:

(Sikora, BÅ‚aszczyk et al. 2013)
The pathway responsible for the hydrogen gas production has been shown in red. Lactic acid bacteria have been found to produce hydrogen in the consortium.
Organic compounds synthesis:

(Peralta-Yahya, Zhang et al. 2012)
Biofuels such as butanol is one of the many organic compounds which can be synthesized using the sludge as the feed of the microbes (Revellame, Hernandez et al. 2012).
Heavy metal detoxifiers:

(Gregoire and Poulain 2014)
Microbes like this phototrophic organism exemplified here are a very valuable source for detoxification of water from heavy metals.
Analytical tests:
Analysis accompanying processes and analytical monitoring of quality parameter:
Industrial water treatment:
fresh water and industrial water treatment,
condensate and feed water treatment,
e.g. analytical monitoring of decarbonization, coagulation, reverse osmosis, desalination, ion exchanger;
Power generation plants and steam generators:
monitoring of water-steam circulations according to statutory regulations (VGB and VdTÜV),
ultrapure water analysis,
flue gas desulphurization, REA-plaster (according to VGB-M 701);
Cooling circuits:
cooling water treatment, cooling water conditioning,
microbiological testing in cooling circuits;
Waste water treatment:
waste water declaration analysis,
control of waste water discharges according to statutory regulations,
supervision of biological waste water treatment plants;
Drinking water analysis, hot water systems (chemically, physical-chemically, microbiologically):
drinking water treatment, distribution networks,
installation of in-house water systems,
water pipe releases;
Ground water analysis:
ground water purification plant,
ground water gauge networks,
landfill leachates;
Check of measuring devices by means of on-site laboratory testing and control testing with
portable testing facilities;
Development of customer-specific solutions and standards for measuring devices;
Waste and residue analysis:
declaration analysis relating to the landfill (LAGA-regulations, TA Abfall),
declaration analysis for the reassembly at the chemical site Leuna;
Composition of foulings in industrial plants.
(Lubello, Gori et al. 2004)
Parameter of water and waste water analysis
pH-value
electrical conductivity temperature
redox potential
oxygen coloration
clouding hardness (total- carbonate- and noncarbonate hardness)
acid and base capacity
permanganate index ((MBAS)
particle size distribution
carbon compounds (TOC, DOC, TIC)
calcite saturation according to DEV C10-R3
nitrogen compounds (TNB
biochemical oxygen demand (5 days) chemical oxygen demand
ammonium
chloride
nitrate[i]
sulfate
nitrite
bromate
sulfide
sulfite
fluoride
settleable solids
filtrate dry residue
test filtratable solids
anionic surfactants
silicic acid
cyanide easily purgeable
iron (total, dissolved, Fe II)
hydrazine
total phosphor
free chlorine
orthophosphate
phenol index
lipophilic substances
nitrification inhibition
depletion test
biodegradability (Zahn-Wellens-test)
suspended solids
References:
Grégoire, D. S. and A. Poulain (2014). “A little bit of light goes a long way: the role of phototrophs on mercury cycling.” Metallomics 6(3): 396-407.
Lubello, C., et al. (2004). “Municipal-treated wastewater reuse for plant nurseries irrigation.” Water Research 38(12): 2939-2947.
Peralta-Yahya, P. P., et al. (2012). “Microbial engineering for the production of advanced biofuels.” Nature 488(7411): 320-328.
Revellame, E. D., et al. (2012). “Lipid storage compounds in raw activated sludge microorganisms for biofuels and oleochemicals production.” RSC Advances 2(5): 2015-2031.
Sikora, A., et al. (2013). “Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence?”.
Describe the entire process for bioinformatics analysis?
Metagenomic analysis of the sludge needs to be done for isolating the useful bacteria and reusing them for the treatment plant. Moreover when this treated water is subjected to reuse then it is necessary to confirm that the disease causing resistant microbes are not present in the water.
First of all the sampling of the sewage needs to be done for micro floral determination. On the basis of biochemical tests the microbes are isolated. For methanogens for example test kits are available article number 01110015 of Vermicon VIT® Methanogenic bacteria; can be used. For hydrogen gas determination fermentation in an airtight container and sampling the overhead air for hydrogen presence is done(Oh, Park et al. 2003). Same goes for the organic synthesis and the enzyme production(Ausec, Zakrzewski et al. 2011).
Phylogenetic analysis of the bacteria e.g. methanogens (Anderson, Ulrich et al. 2009)and others will be done. Their evolutionary characteristics and the genes involved in the biochemical pathway would be studied. For this 16s RNA sequencing will be done and phylogenetic trees will be constructed. This gives us the insight of the microbial pathways and helps us in improving the strains during strain construction and increasing the efficiency of the industrial processes.
After genetics next step is the proteome analysis of the microbes, this is done in metaproteomics, this provides us the functional gene expression information (Schneider and Riedel 2010). As we are using these microbes for useful purposes and commodity generation, therefore we need to have a better understanding whether the genes present in the microbe are functional or not because we have to manipulate them later on. For this purpose 2D gels would be run and the proteins separated can be analyzed by first identifying the sequences, then comparing them with databases. On obtaining the protein information we can easily identify the functional genes of the microbial genome (Wilmes, Wexler et al. 2008).
The useful proteins are the enzymes of the biochemical pathways who are the key players in the product generation. Till here the useful or the productive part of the project has been discussed now the effluent safety needs to be ensured as microbes resistant to the conventional disinfectants need to be identified. (Chao, Ma et al. 2013). For this the resistant genes analysis through metagenome study would be done.
References
Anderson, I., et al. (2009). “Genomic characterization of methanomicrobiales reveals three classes of methanogens.” PloS one 4(6): e5797.
Ausec, L., et al. (2011). “Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes.” PloS one 6(10): e25724.
Chao, Y., et al. (2013). “Metagenomic analysis reveals significant changes of microbial compositions and protective functions during drinking water treatment.” Scientific reports 3.
Oh, Y.-K., et al. (2003). “Isolation of Hydrogen-producing Bacteria from Granular Sludge of an Upflow Anaerobic Sludge Blanket Reactor.” Biotechnology and Bioprocess Engineering 8(1): 54-57.
Schneider, T. and K. Riedel (2010). “Environmental proteomics: analysis of structure and function of microbial communities.” Proteomics 10(4): 785-798.
Wilmes, P., et al. (2008). “Metaproteomics provides functional insight into activated sludge wastewater treatment.” PloS one 3(3): e1778.

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