Determining Concentrations with Spectrophotometer

Determining Concentrations with SpectrophotometerTo learn how to make a spectrophotometer accurately and interpret the data recorded to construct a graph and sustain a cadence curve using excel.To learn how mathematical calculations of the absorbance versions and of unknowns to occur the standard curve of a assimilation appraise from the curve.Method adduce to Proc 2048 Biochemical Engineering Lab Manual employ 1- The SpectrophotometerAbsorbance readings of Methyl orangish and Bormophenol Blue were recorded for a orbital cavity of wavelengths from 400 to 700nm intervals, zeroing the apparatus with a distilled water blank later on severally change in wavelength.Exercise 2 Determination of Glucose ConcentrationEach of the standard glucose solutions and the unknown solutions were tried in spectrophotometer using a wavelength of 580nm and absorbance readings were taken for to each one.Exercise 3- Determination of barm concentrationSimilarly to the glucose experiment absorban ce readings of antithetical standard and unknown barm concentrations were recorded at a wavelength of 600nm. occult solutions U and V were in addition thin to a 12 ratio with distilled water as the concentrations are too high and fall outside the acceptable absorbance range.CalculationsUnknown glucose calculationsy = 0.1836x 0.008, Solve for x givesSubbing in absorbance values for each unknown glucose solution givesUnknown barm calculationsy = 0.557x + 0.003 Solve for x givesSubbing in absorbance values for each unknown glucose solution givesU and V need to be multiplied by 2 after calculation as they were diluted in a 12 dilutionDiscussionIn exercise 1 different wavelengths was used and the absorbance in the graph increasing then decreases and then s well-definedly increasing. The saturation contributes to where the grievous bodily harm absorbance occurs and the concentration affects the intensity of the peaks. For instant the parts blue and the parts colour debile is a green colour. This is the light that we see, and then the wavelengths of light to enlighten through the absorbance with the minimum in terms of all other waves of light absorbance higher.Spectrum analysis of pure sugar solution would be impossible for all absorbance that can happen is the solution to be transparent and any suspend particles. This means that solutions need glucose to the reaction with 1 ml of 3.5 acid Dinitrosalicylic (Domain depict System) to spend a penny amino 3, 5 Nitrosalicylic acid, a compound color absorbs light strongly in all parts of 580nm. This enables us to use phantasmal analysis to determine the rivet. This applies only if the intensity of the color of the product is outright proportional to the concentration of the reactants. In this case, glucose concentration is straightaway proportional to the center of amino 3, 5 Nitrosalicylic acid producers such as the Stoichiometry of the reaction is 101, and most of this focus is not to reach a bal ance It is weighty that the blank or zero concentration used for this experiment is not just distilled water but 1ml of DNS and distilled water made up to the alike(p) volume as the other try ons, as the unreacted DNS in our glucose solutions is contributing to the colour of the solution as well as the 3-Amino,5-Nitrosalicylic Acid.In exercises 3 yeast is basic in terms of chemistry is based on the physics. By increasing the concentration, the absorbance exit increases in yeasts solutions however they are not swarthy but they are block and scatter, so some the light lead not go through them. And this because we are dealing with suspended particulate matter matter, and not resolved ions. Blot out the light commensurate with the focus so that we can find the concentrations of unknown values of absorbance. It is important to shift well before taking the sample absorbance reading such as yeast, particles and settle to the bottom, that mean if we are not shaken them, so they will give us a lower absorbance reading.QuestionsExercise 2The cuvettes wee different surfaces for two primings. The cover ridged sides are so no light escapes out the sides of the cuvette bountiful a false reading. The other reason they have 2 different sides is so that you dont handle the transparent sides directly as oils or whoreson from your fingers could increase the absorbance and give inaccurate resultsParticles in solution (just like in the yeast experiment) affect the absorption reading by blocking or deflecting light away from the detector therefore the particles in a coloured would increase the absorbance and give inaccurate results, unless the concentration and size of the particles is constant with all tests conducted then it would not affect the metric results.A standard curve in spectrophotometric analysis is a analog trendline that fits through your experimental data. It is directd by measuring absorbance readings at a range of different concentrations and plotti ng them against each other. A linear regression do my excel or other means is calculated for the points and an equation in terms of absorbance (Y) and concentration (X) is formed and you can use this equation to calculate unknown concentrations from absorbance readings.Exercise 3The cuvettes have different surfaces for two reasons. The frosted ridged sides are so no light escapes out the sides of the cuvette giving a false reading. The other reason they have 2 different sides is so that you dont handle the transparent sides directly as oils or dirt from your fingers could increase the absorbance and give inaccurate resultsFirstly dilute the dye to an fair to middling concentration with distilled water .Find the maximum absorbance of the diluted dye by testing absorbances at a range of different wavelengths ensuring you zero with distilled between each wavelength. Take some of the dye and dilute it with distilled water to about 6 8 different concentrations i.e. 1100 110, dependin g on what absorbance readings you attempt adjust the dilutions to fit in a range of 0 0.8 as that is where the Beer-Lambert Law applies. Using the max absorption wavelength you would then prepare a standard curve for the dye by measuring absorbance of each of the diluted concentrations. Plot the absorbance vs concentration and use a linear regression to form an equation. Take a sample of the waste water and filter run into any suspended particles to increase the accuracy of the absorbance reading. Finally measure the absorbance of the sample diluting accordingly if absorbance is not in the required range. Using this absorbance value in your standard curve equation calculate the concentration and multiply by your dilution factor if the sample was diluted.Assuming you al immediate have a standard curve and equation for the nitrate complex for a range of 0 1.5 mg/L. All you need to do is dilute your sample from your venture 55 mg/L to fall in the concentration range of 0 1.5 mg/L . So a 150 or a 1100 dilution would be enough to give you 1.1 mg/L or 0.55 mg/L respectively. Then the sample is ready for spectrophotometer analysis.ConclusionAll of the yeast and sugar were obtaining accurate test results with the value of R2 (0.99) for each of the trend lines and graphs. 99% accuracy meaning that lines fits for most the points. The turn was a good knowledge to learn and it is useful to understand the Beer-Lambert legality and his applications.