Thursday, April 4, 2019
UV Visible Spectrophotometry and Solution Absorption
UV Visible Spectrophotometry and Solution Absorption tout ensemble bulwarkecules absorb shed light on at certain wavelengths. The denseness of light by a firmness of purpose whitethorn be used to meet the tautness of a solute or a mixture of solutes in solution.The Beer-Lambert lawfulness refers to the linear relationship between absorbance (A), and assiduity (C) of an absorbing species. correspond to the two fundamental principals that govern the immersion of light by a solution, the density of light passing through a solution is exponentially related to the number of molecules of the absorbing solute, and thus the solute intentness, and the length of the absorbing solution.These principals argon combined, and when working in ducking units of molarity, the Beer-Lambert law is as followsFor part A of this experiment the value at the pocket for Vitamin B12 was coiffed by measuring the absorbance of a known immersion of Vitamin B12 and by employ the preceding(prenomina l) Beer-Lambert mandate. Vitamin B12 is a compound of profound nutritional and clinical importance. Assaying and understanding absorption of vitamin B12 helps with diagnosis of defects in humans that brush aside lead to hematological and neurological complications.For part B of this experiment chlorophyll compactness of a finger condense was cipher. In context to the experiment, eukaryotic green plants and algae, and prokaryotic cyanobacteria contain chloroplasts which turn in several paint types, the about abundant of these being chlorophyll a.Green and grim-green coloured chlorophyll a absorbs soapimum light energy at the photosynthetic reaction centre (during the light reaction of photosynthesis) at wavelengths in the blue ( scoop shovel 420 nm) and red (max 663 nm ) regions of the visible spectrum.The green-yellow coloured chlorophyll b is also accede in all green plants and has an absorption spectrum (red max 645 nm and blue max 435 nm) slightly disparate from chl orophyll a.Normally the balance of chlorophyll ab is 31. 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 altered into chlorophylls a and b.The amounts of chlorophyll and other pigments in plants bath be determined use a spectrophotometer following downslope with various organic resolving powers.Based on the Beer-Lambert Law and a association of absorption coefficients of pigments dissolved in particular solvents, equations have been derived to right away determine the concent dimensionns of common pigments following extraction by measurement of the absorbance (A) of the solution at a buildn wavelength (max) in a cuvette.For part 3 of the experiment, protein assiduity was determined by use of UV and Visible spectrophotometry, and Construction of a measure Graph.The approximation of protein concentration is an important measurem ent in biological sciences. For pure archetypes of proteins absorbance measurements at 280 nm can be used to signally determine protein concentration all proteins absorb in this region of the spectrum due to their redolent(p) amino acid differences (tyrosine, tryptophan and phenylalanine).For protein mixtures, very dilute solutions, or for proteins with interfering chromophores, colourimetric method actings essential be used. These assume overmatching a pure protein warning of known concentration to a colourimetric reaction, and measuring the absorbance of the coloured end product. The consume protein of unfamiliar concentration is subject to the same colourimetric reaction. The concentration of the sample protein can be read civilisely 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 deter mination of the concentration of lysozyme in solution. The first of the methods makes use of a max in the UV part of the spectrum and the other in the visible part of the spectrum.AimsTo competently use a spectrophotometer and accociated cuvettes (cells)To relate absorbance of a solution to concentration employ the Beer-Lambert lawTo determine the molar absorption (extinction) coefficient of vitamin B12 and compare its value with that from a standard reference point table.To calculate the chlorophyll concentration in a flip-flop extract use 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.Methods mathematical function ATo begin the experiment, the spectrophotomer was calibrated in accordance to the information given in the instrumentation booklet (p. 35, viii). apply distilled water in a plastic cuvette at a wavelength of 550 nm the spectrometer was because pose on zero. using the provided Aqueous Vitamin B12 (cyanocobalamin) solution at a stock concentration of 0.15 g dm-3 (relative molecular(a) mass = 1.355 x 103 i.e. 1,355 Daltons ), The A value was measured and recorded at max 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 value for Vitamin B12 was calculated (see computer sciences).Part BFor 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 devolve pigment solution was needed for the test and so a check was carried out to ensure that in that respect 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 cuv ette. The spectrophotometer was placed on zero using a quartz cuvette filled with an aqueous acetone mixture (80%) ready 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 common chord sets of data were recorded.Part C(a) Direct absorbanceA 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 max of 280 nm. The value obtained was recorded.Having measured the A280 value of the unknown lysozyme sample, the concentration of lysozyme was ca lculated taking into consideration that 280 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 ( conceptualisation 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 to a lower placeTube No12345678BSA stock(cm3)1.01.52.02.53.03.54.05.0 weewee (cm3)4.03.53.02.52.01.51.00.0Note that the dilution factors for each supply were used to enable calculations for final concentrations of BSA in pipages 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 L owry C ( alkalic 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 Ciocalteus phenol reagent) was then made up by diluting Folin reagent (5 cm3) with distilled H2O (10 cm3).Lowry C reagent (5.0 cm3) was added to all ten test tubes. The solution was mixed and left for 10 legal proceeding. Lowry D reagent (1.0 cm3 ) was then added to each test tube and mixed well. All tubes were left for 30 minutes at standard temperature (37oc) for reaction and colour development to occur, after which time the test tube table of contentednesss were thoroughly mixed.For test tubes 1-9, the A value at max 750 nm was measured. Test tube 10 was not measured as it was used as a H2O/reagent blank to zero the spectrophotometer.CalculationsPart ADue to the fact that a known amount of solute has to be dissolved in a given volume of solvent to obtain a solution of the required concentration, the number of moles of the solid ca n be calculated from the following equationn = Mass of soluteRelative molecular massTo convert the Aqueous Vitamin B12 (cyanocobalamin) solution from g dm -3 to mol dm-3 one must consider that the stock concentration is 0.15 g dm-3, and the relative molecular mass of Vitamin B12 is 1.355 x 103 .0.15 / 1.355 x 103 = 0.11 x 103To find the value (wavelength absorption coefficient) of vitamin B12 the Beer-Lambert law must be appliedA= 0.827L= 1cmC= 0.11 x 103 dm3 mol-1 cm-1 at max of 550 nm = unknownAs A= LC, the equation can be rearranged as follows to make the subject = A/CTherefore = 0.827/0.11 x 103 = 7.51 x 103Part B chlorophyl assiduity determinationThe following formula was used to calculate the concentration of pigment in the extract.chlorophyll a = 12.67A663 2.65A645 0.29A626Chlorophyll b = 23.6A645 4.23A663 0.33A626Protochlorophyllide = 29.6A626 3.99A663 6.76A645The absorbance (A) is the respective wavelengths obtained directly from the spectrophotometer with the use of a 1cm light path length cuvette.Chlorophyll a = (12.67 x 0.934) (2.65 x 0.390) (0.29 x 0.321) = 10.71 ug cm-3.Chlorophyll b = (23.6 x 0.934) (4.23 x 0.390) (0.33 x 0.321) = 20.29 ug cm-3.Protochlorophyllide = (29.6 x 0.934) (3.99 x 0.390) (6.76 x 0.321) = 23.92 ug cm-3.Part C(a) Direct absorbanceConcentration of lysozyme was calculated using the Beer-Lambert law as followsA = 0.177 = 3.65 x 104 dm3 mol-1 cm -1L = 1cmC = UnknownThe Beer-Lambert law can be rearranged, qualification C the subject of the equation. Therefore the value of C can be calculated asC = A / C = 0.177 / 3.65 x 104 = 4.84 x 10-6 mol dm-3The concentration of the lysozyme sample was then changed from mol dm-3 to gcm-3Using the following formulan= M / RMM14.31 x 103 x 4.84 x 10-6 = 0.069gTo change this from g to g it must be multiplied by 1000,000 as follows0.069 x 1000,000 = 69000To then change this calculation from dm-3 to cm-3 it must be divided by 1000 as follows69000 / 1000 = 69 g cm-3(b). Preparation and Use of a Standard Curve for Lowry AssayConcentration (g cm-3) was calculated using the to a lower place figuresTube No12345678BSA stock(cm3)1.01.52.02.53.03.54.05.0H2O (cm3)4.03.53.02.52.01.51.00.0Test tube 1.BSA stock (cm3) = 1.0H2O (cm3) = 4.01.0 + 4.0 = 51.0 / 5 = 0.20.2 x 250 = 50Test tube 2.BSA stock (cm3) = 1.5H2O (cm3) = 3.51.5 + 3.5 = 51.5/5 = 0.30.3 x 250 = 75Test tube 3.BSA stock (cm3) = 2.0H2O (cm3) = 3.02.0 + 3.0 = 52.0 / 5 = 0.40.4 x 250 = 100Test tube 4.BSA stock (cm3) = 2.5H2O (cm3) = 2.52.5 + 2.5 = 52.5 / 5 = 0.50.5 x 250 = 125Test tube 5.BSA stock (cm3) = 3.0H2O (cm3) = 2.03.0 + 2.0 = 54.0 / 5 = 0.60.6 x 250 = 150Test tube 6.BSA stock (cm3) = 3.5H2O (cm3) = 1.53.5 + 1.5 = 53.5 / 5 = 0.70.7 x 250 = one hundred seventy-fiveTest tube 7.BSA stock (cm3) = 4.0H2O (cm3) = 1.04.0 + 1.0 = 55.0 / 5 = 0.80.8 x 250 = 200Test tube 8.BSA stock (cm3) = 5.0H2O (cm3) = 0.05.0 + 0.0 = 55.0 / 5 = 11 x 250 = 250Results1. Molar absorption coefficient of vitamin B12Absorbance ren dering at max of 550nm (A550nm) 0.8272. AbsorptionPigment typeA ValueAbsorptionConcentrationChlorophyll aA663nm0.93410.71Chlorophyll bA645nm0.39020.29ProtochlorophyllideA626nm0.32123.92Fig. 1. a table bespeaking the A value of deuce-ace unalike pigment types found in a leaf extraction, and the measured absorbance and calculated concentration of each.3. Lysozyme Concentration Determination(a) Direct absorbance reading at max of 280 nm (A280nm) 0.177(b). Preparation and Use of a Standard Curve for Lowry AssayDiscussionPart AA). Using the data collected, the experimental the value (dm3 mol-1 cm-1) calculated during this experiment was compared to that of an value obtained fromcommercial standard references data.(nm)(dm3 mol-1 cm-1)Standard value550nm8.55 x 103Experimental value550nm7.51 x 103Fig. 4. a table to show a comparison between standard and experimental values.As the above data indicates, the experimental value obtained during this experiment differs from that of the standard value.B). Other than human and experimental errors, one surmisal that could explain the differences in the values is that to a certain degree dissimilar spectrophotometers in the laboratory give different readings. If the standard value was recorded using a different spectrophotometer this could cause anomalies within the results.A second possibility is that the solution used to find the experimental value was not at a stock concentration of exactly 0.15 g. This would affect the calculations and hence a different result would be obtained to that of the standard value.Part B1(a).The % of chlorophyll a, chlorophyll b and protochlorophyllide in the leaf extract can be calculated in the following waychlorophyll a concentration = 10.71 ug cm-3chlorophyll b concentration = 20.29 ug cm-3protochlorophyllide concentration = 23.92 ug cm-310.71 + 20.29 + 23.92 = 54.92Percentage of chlorophyll a = 10.71/54.92 x 100 = 19.5%Percentage of chlorophyll b = 20.29/54.92 x 100 = 36.9%Pe rcentage of protochlorophyllide = 23.92/54.92 x100 = 43.6%1(b).It can be seen from the above percentages that 43.6% of the leaf extract is composed of protochlorophyllide. This is the largest percentage play within the leaf extract and therefore it can be considered as the predominant pigment type.2(a). The percentages obtained can also be used to calculate the ratio of chlorophyll ab in the leaf extract. In the case of this experiment the ratio of chlorophyll ab in the leaf extract was 21.2(b). The determined value to be expected when calculating the ratio of chlorophyll ab in the leaf extract was 31. One would expect this as there are three pigments in the leaf extract, that ideally should contri only whene evenly. However, The results from this experiment vary from the standard data as they show a ratio of 21 between chlorophyll a and b.The most probable author for this variation is not due to anomalies in results or calculations, but the fact that not all leaf extracts will co ntain the standard amount of pigments. In some cases pigments may be present in a higher percentage of one than the other, as is this experiment where protochlorophyllide was the predominant pigment type.3). Although the above ratio is only dealing with percentages of chlorophyll a and b present in the leaf extract, one must consider that the presence of protochlorophyllide must be allowed for in the formulae and hence in the calculations. This is because there are three pigment types involved in the leaf extract and so one must consider that the ratio of the whole leaf extract is actually 321 where the ratio of chlorophyll ab is 21.4). The use of a formula related to a given extraction solvent is a convenient method for determining the concentration of chlorophyll. However, making use of the Beer-Lambert Law the concentration of chlorophyll could also be found by preparation and use of a standard curve.Part CDetermination of Protein Concentration by UV and Visible Spectrophotometry , Construction of a Standard GraphProteins have aromatic side chains such as tryptophan tyrosine and phenylalanine which absorb light at 280nm.The Lowry method is based upon a combination of the biuret method and the oxidation of tyrosine and tryptophan residues.The biuret reaction involves the binding of Cu2+ under alkaline conditions to nitrogen found in the peptide bonds of proteins. This reaction gives off a deep blue colour. The folin reagent contains phosphomolybdotungstate acids which are trim down to tyrosine, tryptophan and polar amino acids. This creates an intense blue-green colour.(a). The data collected was used to create a graph, plotting a standard curve of A (Y axis) against BSA concentration (X axis) in g cm-3 (See fig.3). This graph, shown in fig.3., was effectively constructed anticipate that the relationship between absorbance (A) and concentration (C) must be linear to satisfy the Beer-Lambert law. However, the Beer-Lambert relationship between absorption an d concentration deviates from lineariy in the case of more concentrated solutions.Linear BSA standard curves are only obtained at low protein concentration and so therefore to decrease possible anomilies in the results, timing of both residue addition and mixing were crucial.Using the A value from test tube 9, it was possible to use the graph to determine the concentration of the unknown lysozyme sample in g cm-3. Results from the graph show that.(b) By examining the lysozyme concentration results obtained (in g cm-3), it is possible to make a comparison between the results for the colourimetric assay and the direct absorption technique.Results show that Lysozyme concentration for colormetric assay were (m/rmm thing)Lysozyme concentration results for direct absorbance technique were..(graph)These results are same/different.Due to the fact that different proteins have unsubtlely varying characteristics, there may be considerable errors within the data. With the colormetric assay any non-protein component of the solution that absorbs UV light could interfere with the assay, resulting in the production of colour by substances other than the analyte of interest. This would cause the results to vary from that of the direct absorbance technique.(c). For this experiment three different methods were used for concentration determination, each of which had different strengths and weaknesses with respect to their sensitivity, accuracy and convenience.The first of these methods was the use of a formula, to determine chlorophyll concentration.Using a formula gives a very accurate theoretical result but it is not particularly convenient as for calculations to be correct it can take a bully deal of time and effort. Obviously with such calculations, they are not sensitive as there is no outside interference to affect results.Direct absorbance is not as sensitive as the colormetric method, but as it requires the use of a spectrophotometer, it is an accurate assay method. This also makes the method relatively convenient for determining the concentration of lysozyme present in a given solution as changes in absorbance of the lysozyme could be clearly seen and recorded using the spectrophotometer at a particular wavelength.The colourimetric method was also used to determine the concentration of lysozyme during this experiment. One benefit of using the Colormetric method is that it is extremely sensitive (down to a protein content of 20ug ml-1) and it is also moderately constant from one protein to another. However, with respect to accuracy, this method is subject to interference from a wide range of non-protein substances including many organic buffers. The choice of an appropriate standard is important as the intesnsity of colour produced for a particular protein is dependant on the number of aromatic proteins.As different proteins have a different number of aromatic residues, the Lowry assay is considered more of a qualitative measure of protein content more than denary method of determining protein concentraion.This method is not as convenient as the direct absorbance method in that it takes a lot longer to perform and there is a higher frequency of anomalies that must be accounted for.(d). The measurement of protein levels is of significant diagnostic importance in both clinical and veterinary medicine. In clinical medicine there are a wide variety of biomedical tests involving the measurement of protein levels, such as the detection of abnormal protein levels in cerebrospinal fluid (CSF), suggesting that there is an abnormal process occurring in the rally nervous system.Protein levels in urine samples are tested to monitor and evaluate kidney function, and essentially to detect and cite kidney damage and disease at and early stage. Serum protein tests are also important as they foreboding measurement of protein levels of albumin and globulin in the blood.Such tests are also important in veterinary medicine. According to repo rts from Cornell universities college of veterinary medicine, protein tests have been developed to accurately indicate canine liver failure caused by the toxin aflatoxin.(e). other way in which protein concentration can be measured, other than by the use of a formula or a spectrophotometer is gel electrophoreses. This technique uses charged protein molecules to separate physical properties, as they are oblige through a gel by an electrical current.