A Laboratory Report On Spectrophotometry

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

Perform a series of accurate tests on biological molecules to detect the presence of carbohydrates and proteins, as well as the action of an enzyme on specific molecules.

During this experiment, the activity of catechol oxidase and the absorbance of benzoquinone was measured in five solutions with different pH concentration. Five cuvettes provided by the instructor were used during the experiment. The transmittance on the spectrophotometer was zeroed on an empty chamber and it was set at a wavelength of 486 nm. Because the shape of an enzyme changes with different pH concentration and each pH has different chemicals, so five cuvettes containing 0.5 mL of enzyme solution and 4.5 mL of pH 2, pH 6, pH 7, pH 8, pH 11 were used to make five different blank solutions (Scott, S.M. et al 2016). (Each blank was made when needed, not all blanks were made at the same time.)

The image above is our chromatogram, you can see at the top the orange bands of a carotene, next are the bands of xanthophylls which are yellow. The next light green bands directly bellow the yellow is chlorophyll a, the darker green bands are chlorophyll b. In order to test absorbance of the chlorophylls they were cut out of this chromatogram submerged into acetone and then used to get an absorption spectrum as seen below.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

A spectrophotometer’s purpose is to use colors of the light spectrum to determine the concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission was to determine the protein concentration and the standard curve of the unknown sample of BSA. This, by preparing five dilutions of the unknown solution of BSA together with other known concentrations, and then experimenting by observing how the concentrations were passed through the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this

Scientists use an instrument called a spectrometer to quantitatively determine the amount of light absorbed by a solution. The primary inner parts of a typical spectrometer are described below. The spectrometer has a light source that emits white light containing a vast mixture of different wavelengths of electromagnetic radiation. The wavelength of interest is then selected using a monochromator (“mono” meaning one and “chromate” meaning color) and an additional exit slit. The separation of white light into different colors (wavelengths) is known as diffraction. The selected light then reaches the sample and depending on how the light interacts with the chemical compound of interest, some of the light is absorbed and some passes straight through. By comparing the amount of light entering the sample (P0) with the amount of light reaching the detector (P), the spectrometer is able to tell how much light is absorbed by the sample.

6. A spectrophotometer would definitely be useful in experiments involving the measurement of concentration that produces color, since the color absorbance can be used to find concentration. One experiment involving a spectrophotometer could be finding the molarity of a blue Copper(II) sulfate solution. The wavelength could be set to red, and then the absorbance values can be correlated to the molarity of Copper(II)

When you use a spectrophotometer you should not set the wavelength of light to be the same color of the solution. This is because if you set the wavelength to be the same color of the solution then no light will be absorbed. The reason why no light will be absorbed is because the color you see is the wavelength of light that is being reflected so you must set the wavelength to be the complementary color. The wavelength of light that was chosen for the lab was 450nm which coincides with a very dark blue color. The reason why choosing a dark blue makes the most sense for this experiment is because the color of the FeSCN2+ ion was blood red.

Using the yellow tube, which included everything but starch, as the blank, each group zeroed their spectrophotometer. This was done so that any absorbance observed depends only on the amount of starch present, not on any other reagents (buffer, IKI). To zero the spectrophotometer, the wavelength was first set at 580nm, using knob 3 (45). Next, the groups made sure that the light next to “transmittance” was lit, and the chamber to be tightly closed. Having the chamber empty & closed tightly provides reference for the darkest condition possible. Using knob 1, the transmittance was turned until it read 0.0 (45). Before the groups used their blank test tube to zero the spectrophotometer, each needed to wipe the tube with kimwipes to ensure a clean reading. Turning knob 2, each group was then instructed to zero the absorbance, 0.000. Upon removing the blank, each trial was inserted into the chamber (46). The

The dyes in the laboratory experiment are made of numerous colors, mainly red and blue, the spectra from each of the dyes corresponded to the wavelengths obtained from each of dye i.e. 620 nm for red and 450 nm for blue.

The spectrophotometer was turned on 15 min prior to the experiment. For each different intensity levels, 2 cuvettes were prepared (one for the experimental variable and one for the control variable). Also, 2 cuvettes were prepared for each different wavelength. Two blanks were prepared for the entire experiment (one for intensity, one for wavelength). The experimental cuvette for intensity consisted of 2.5 ml of 2.5 ml DCPIP, 2.0 ml water, 2.0 ml PO4 buffer, and 0.2 ml chloroplasts, a total of 6.7 ml. The control cuvette for intensity was the same as the experimental cuvette for intensity. The experimental cuvette for wavelength consisted of 2.5 ml DCPIP, 1.7 ml water, 2.0 ml PO4 buffer, and 0.5 ml chloroplasts a total of 6.7 ml. The blank cuvette intensity contained 4.5 ml of water, 2.0 ml of PO4 buffer, and 0.2 ml chloroplasts for a 6.7 ml. The wavelength blank was composed of 4.2 ml water, 2.0 ml PO4 buffer, and 0.5 ml chloroplasts for a 6.7 ml. Before starting the experiment, we set the wavelength at 600 nm, placed the blank cuvette into the spectrophotometer, and set the absorbance at zero. The laboratory was kept dark during the experiment to prevent light pollution.

The predictions that were made prior to the experiment being ran about the concentration of pigments in Drosophila were similar to the observed concentration of pigments. Once four fly heads of each pigment were crushed onto filter paper, chromatography was ran. After six hours of the chromatography being run, they were removed from the solvent and dried. Once the filter paper was dry, it was placed under a UV light for a better observation of the pigments present in the eyes of the fly. What was found in the white was much less than in the wild type due to the fact that in the white eyed fly, both enzymes that start the pigment production (the pteridine pathway and the ommochrome pathway) are blocked or changed so no pigments can be produced.

Spectrophotometry is a routine laboratory test that has the added advantage of being able to

In this experiment, the use of a glass cuvette was used in order to avoid any chemical or environmental interference from the surroundings. Furthermore such glass cuvettes can support large wavelengths during transmission of light beyond 320 nm since this is the excitation wavelength region. Riboflavin is excited at around 370 nm, therefore the wavelength range must be large in order to detect a proper response. Plastic cuvettes are however inadequate as the range of wavelengths supported for transmission of light is very limited (5, Upstone). The geometry of the instrument of the spectrophotofluorometer has a specific angle and position for the excitation source and the detector. The excitation polychromator emits ultra-violet radiation and light to the interested sample and the shape of the polychromator resembles a triangular prism in which the monochromator has been angled at a 90∘ position. The transducer would detect the irradiation excitation beam and transfers it through the emission polychromator. The emitted beam would then travel to the detector to allow for the response signal to appear for the sample on the computer system (416, Skoog). The position of the cuvette is essential as it enables for the consistent readings of the response signals, as any shift in the cuvette position can create variation in results due to the position of lighting and detection of molecular samples hitting at different angles. The process of fluorescence is quite sensitive than absorption due to having lower detection limits because of the fact that it can detect lone molecules in excited states that are irradiated by UV-light unless they come into contact with other molecules within