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UV Visible Spectrophotometry and Solution Absorption

All molecules absorb light at certain wavelengths. The absorption of light by a solution may be used to determine the concentration of a solute or a mixture of solutes in solution.
The Beer-Lambert law refers to the linear relationship between absorbance (A), and concentration (C) of an absorbing species.
According to the two fundamental principals that govern the absorption of light by a solution, the absorption of light passing through a solution is exponentially related to the number of molecules of the absorbing solute, and thus the solute concentration, and the length of the absorbing solution.
These principals are combined, and when working in concentration units of molarity, the Beer-Lambert law is as follows:
For part A of this experiment the ε value at the max for Vitamin B12 was determined by measuring the absorbance of a known concentration of Vitamin B12 and by using the above Beer-Lambert formula. Vitamin B12 is a compound of significant nutritional and clinical importance. Assaying and understanding absorption of vitamin B12 helps with diagnosis of defects in humans that can lead to hematological and neurological complications.
For part B of this experiment chlorophyll concentration of a leaf extract was calculated. In context to the experiment, eukaryotic green plants and algae, and prokaryotic cyanobacteria contain chloroplasts which have several pigment types, the most abundant of these being chlorophyll a.
Green and blue-green coloured chlorophyll a absorbs maximum light energy at the photosynthetic reaction centre (during the light reaction of photosynthesis) at wavelengths in the blue (max 420 nm) and red (max 663 nm ) regions of the visible spectrum.
The green-yellow coloured chlorophyll b is also present in all green plants and has an absorption spectrum (red max 645 nm and blue max 435 nm) slightly different from chlorophyll a.
Normally the ratio of chlorophyll a:b is 3:1. As with most biological molecules chlorophyll is synthesised by biochemical pathways, and one intermediate molecule in the synthesis pathway is protochlorophyllide (max 626 nm) which is eventually converted into chlorophylls a and b.
The amounts of chlorophyll and other pigments in plants can be determined using a spectrophotometer following extraction with various organic solvents.
Based on the Beer-Lambert Law and a knowledge of absorption coefficients of pigments dissolved in particular solvents, equations have been derived to directly determine the concentrations of common pigments following extraction by measurement of the absorbance (A) of the solution at a given wavelength (max) in a cuvette.
For part 3 of the experiment, protein concentration was determined by use of UV and Visible spectrophotometry, and Construction of a Standard Graph.
The estimation of protein concentration is an important measurement in biological sciences. For pure samples of proteins absorbance measurements at 280 nm can be used to directly determine protein concentration; all proteins absorb in this region of the spectrum due to their aromatic amino acid residues (tyrosine, tryptophan and phenylalanine).
For protein mixtures, very dilute solutions, or for proteins with interfering chromophores, colourimetric methods must be used. These involve subjecting a pure protein standard of known concentration to a colourimetric reaction, and measuring the absorbance of the coloured end product. The sample protein of unknown concentration is subject to the same colourimetric reaction. The concentration of the sample protein can be read directly from a standard curve.
The Lowry assay involves the production of a blue (phosphomolybdate-tungstate) chromophore, from a copper-protein complex.
In this part of the practical, Lowry and direct absorbance methods were compared for the determination of the concentration of lysozyme in solution. The first of the methods makes use of a λmax in the UV part of the spectrum and the other in the visible part of the spectrum.
To competently use a spectrophotometer and accociated cuvettes (cells)
To relate absorbance of a solution to concentration using the Beer-Lambert law
To determine the molar absorption (extinction) coefficient of vitamin B12 and compare its value with that from a standard reference table.
To calculate the chlorophyll concentration in a leaf extract using absorbance values at defined wavelengths and a formula applicable to the solvent extraction medium.
To measure protein concentration using direct absorbance and, following construction of a calibration curve, by a colourimetric method.
Part A To begin the experiment, the spectrophotomer was calibrated in accordance to the information given in the instrumentation booklet (p. 35, viii). Using distilled water in a plastic cuvette at a wavelength of 550 nm the spectrometer was then placed on zero.
Using the provided Aqueous Vitamin B12 (cyanocobalamin) solution at a stock concentration of 0.15 g dm-3 (relative molecular mass = 1.355 x 103 i.e. 1,355 Daltons ), The A value was measured and recorded at λmax at 550 nm. The A value was Placed on the results sheet.
The vitamin B12 solution concentration was converted from g dm -3 to mol dm-3 and then using this data the ε value for Vitamin B12 was calculated (see calculations).
Part B For the second part of the experiment a sample of pigments extracted from dandelion leaves homogenized in an aqueous acetone extraction medium (80%) was provided. A clear pigment solution was needed for the test and so a check was carried out to ensure that there was no plant debris that may have interfered with light passage before the absorbance of the sample was measured.
Using a Pasteur pipette, the clear extract was transferred into a clean quartz cuvette. The spectrophotometer was placed on zero using a quartz cuvette filled with an aqueous acetone mixture (80%) set at a max wavelength of 663 nm and the absorbance of the pigment solution was measured at 663 nm.
The spectrophotometer was again placed on zero using the acetone solution (80%), however it was set at a max wavelength of 645 nm before the absorbance of the pigment solution was measured.
The spectrometer was placed on zero for a third time and set at max wavelength of 626 nm. The absorbance of the pigment solution was again measured and all three sets of data were recorded.
Part C (a) Direct absorbance A quartz cuvette was filled to the level with H20 and used as a standard to set the spectrophotometer at zero. Using another quartz cuvette the A value of the lysozyme solution of “unknown” concentration was measured at a λmax of 280 nm. The value obtained was recorded.
Having measured the A280 value of the “unknown” lysozyme sample, the concentration of lysozyme was calculated taking into consideration that ε280 of lysozyme = 3.65 x 104 dm3 mol-1 cm-1 and using the Beer-Lambert Law. The concentration of the lysozyme sample was then changed from mol dm-3 to gcm-3.
(b) Colourimetric Lowry Assay (Preparation and Use of a Standard Curve)
Using a stock reference standard BSA solution containing 250 g cm-3 protein, a series of dilutions of the stock were prepared accurately, as per the table below:
Tube No:
BSA stock
H2O (cm3)
Note that the dilution factors for each tube were used to enable calculations for final concentrations of BSA in tubes 1- 8 inclusive (see calculations). These values are then used to plot a standard curve.
Standard solution (1.0 cm3) prepared in the above table was placed in 8 clean, dry test tubes. “unknown” lysozome sample (1cm3) was placed into test tube 9, and H2O (1.0 cm3) was placed in test tube 10 as a water/reagent blank control.
A solution of “Lowry C” (alkaline copper reagent) was made up by mixing “Lowry B1” (0.5 cm3) with “Lowry B2” (0.5 cm3) and “lowry A” (50 cm3). A solution of “lowry D” (Folin

Bad Odor and Bio-filtration Solutions

Mikhail Kachmazov
UQ: How have microbes solved a local or global problem? The specific problem or issue:
Sewage and industrial plants situated near residential areas can produce unpleasant odors making it difficult to live there.
Explain the problem:
The sewage plants near residential areas can be a subject to the social and environmental problems if these facilities produce unpleasant odors. The causes of the odors are generally the inorganic and volatile organic compounds which result from bio filtration and from the sewer of industrial waste. There are different types of volatile organic compounds that are emitted as a result of bio filtration like 2-butanone, α-pinene, tetrachloroethylene, dimethyl disulfide, β-pinene, limonene, phenol and benzoic acid. One of the main culprits for the bad odor are sulphur compounds which are relatively less in concentration but play significant role in odor causing factors.[5]
Explain how science is helping, or has helped, to solve the problem:
Bad odor from sewage water is a major environmental issue worldwide. Bad odor from sewage water is an indicator of possible health risk therefore governments spend a lot of money on treating the sewage water and unpleasant odor. Science has played a major role in solving this problem biologically as well as chemically. Following are several methods to treat sewage water and odor from this water.
Bio filtration
Thermal Oxidation
Thermal oxidation burns the odor causing compounds directly or indirectly. Bio filtration on the other hand oxidizes the odor causing compounds by using microbes. Microbes like Pseudomonas Putida that are used in this technique have been significant in treating sewage water and bad odor in an efficient, safe and inexpensive way. [4]
How it works:
Bio-filtration is quite an innovative technology to control pollutants. It helps to eliminate malodorous gas emissions and volatile organic compounds (VOCs) of low concentrations. The most common design of a bio-filter is just an ordinary big box. Some of them can be very big, others can be quite small. A bio-filter’s main function is to bring microorganisms together with pollutants in an air stream. The bio-filter which has the breeding material for the microorganisms is placed inside the box. The “biofilm”, which is a layer of moisture where the microorganisms live, can be found around the particles of filter media.

When the bio-filtration process takes place, the operators pump the polluted stream of air through the bio-filter, so that the filter media absorbs the pollutants. The bio-filter diffuses the contaminated gas and sends it onto the biofilm that absorbs it. The pollutants are then degraded by the microorganisms. The metabolic products of this process are carbon dioxide and water as well as the produced energy. The chemical formula of this process caused by oxidation is:
Volatile Organic Pollutant O2ïƒ CO2 H2O Heat Microbial Biomass [3]
Effectiveness of this technique:
This technique is very efficient and it has plenty of advantages over the traditional methods of pollutants. First of all, one of the major conditions for traditional methods is high temperature, however when it comes to bio-filtration the technicians can use low temperatures, thus the whole process becomes cost effective, because there are no costs of combustion. Moreover it is safer than traditional methods because combustion is a dangerous process. Secondly, the maintenance cost of bio-filtration is much lower than traditional methods. According to a research conducted by Pinchin Environmental Group Canada, this technology is more than 95% efficient and environmentally friendly because the whole process can be done in an absolutely natural way. [1]
Limitations of this technique:
Even though this is an effective method it still has some limitations that restrict the implementation of this technique. First of all the bio-filtration is not able to remove all the organic substances from the water, for instance, those with low rates adsorption or degradation like chlorinated VOCs. Moreover this technique requires a large area to be installed in case it will need to treat a large amount of water. Finally, acclimation periods for populations of microbes may be as long as weeks or months, in particular for volatile organic compounds treatment.
Applications of Bio-filtration:
Even though the simple forms of bio-filters can be used by general public, the main uses are in the commercial areas. The following industries apply VOC:
Chemical and petrochemical industry
Synthetic resins
Paint production
Oil and gas industry
Pharmaceutical industry
Treatment of wastewater
Remediation of Soil and Groundwater
The following industries apply odor abatement applications:
Treatment of sewage
Slaughter houses
Gelatin and glue plants
Agricultural processing
Tobacco, sugar and cocoa industry
Fragrance and flavor
Environmental, health and social impact:
This technique is environmentally friendly because it does not damage the environment and it does not pollute the air or anything. It’s eco-friendly because the process is natural and does not harm the nature of the Earth, it is also been called a “green technology”. Moreover, it has positive health impact on the society, because the process of bio-filtration does not involve any chemical substances and does not produce any harmful chemical by-products. Basically, the only by-products of the process of oxidation used in bio-filtration are water and carbon dioxide, which makes this technology extremely clean and safe.
To conclude I want to say that the bio-filtration process is an effective process that is used frequently nowadays. Even though it has its own limitations and disadvantages, still it is one of the most promising, environmentally friendly and efficient technologies, that combines both simplicity and effectiveness, therefore it is a viable alternative to traditional methods of sewage water treatment.
“Odour Complaints.” Pinchin Environmental. N.p., n.d. Web. 08 Feb. 2014.
“Pseudomonas Putida.” Pseudomonas Putida. Kris Hamilton, n.d. Web. 06 Feb. 2014.
“Sewage and Wastewater Odor Control” by Dr. Giancarlo Riva, Ozono Elettronica Internazionale, Muggio, Italy Anthony Sacco, Spartan Environmental Technologies, Mentor, OH, USA Treatment/Papers/Technical Papers/Municipal-Odor-Control-Italy TP.pdf
“Detection, Composition and Treatment of Volatile Organic Compounds from Waste Treatment Plants” by Xavier Font, Adriana Artola and Antoni Sánchez , Composting Research Group, Department of Chemical Engineering, Universitat Autònoma de Barcelona, 08193-Bellaterra (Cerdanyola del Vallès), Barcelona, Spain.