Spectrophotometry

This experiment was used to measure the buildup of the colored product, benzoquinone, to observe the change in the absorbance of the mixture in a spectrophotometer at a wavelength of 486 nm. It was hypothesized that over a longer period catechol oxidase activity and reaction in the mixture will continue, creating more benzoquinone resulting in an increase in the absorbance. The hypothesis was supported by the data as seen in figure 2 which shows the positive relationship between time and absorbance. As can be seen, with more time the catechol oxidase can catalyze the reaction and turn catechol (substrate) into benzoquinone (product). This in turn increased absorbance of the blue light at 486 nm by the solution containing benzoquinone which has a dark brown

• Secondly, we also used a colorimeter which is extremely accurate when it comes to measuring the percentage light transmission. As it measures the percentage light transmission as a numerical value.

From this graph and chart we can see that the higher the concentration the higher the absorbance, all the different concentrations were tested at the same wavelength (625nm). Also we can determine our unknown substances concentration by using the absorbance we got for it. The red dot on the graph followed by the line towards the horizontal axis indicates that the concentration of fast green was 34% or 5.1x10-3.

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

Time) because it had a correlation closest to 1. All three orders were graphed and a linear regression was used to see which graphed order was closest to 1. The order was determined by comparing the concentration and time to the mathematical predictions made using the integrated rate laws. Analyzing each graph and finding each correlation helped determine which graph was closest to 1. The more concentrated a solution is, the higher the absorbance of that solution. This is due to Beer’s Law. The law measures the absorbance of a solution by determining how much light passes through a solution. As the concentration of a solution increases, fewer wavelengths of light are able to pass through the concentrated solution. The absorbance at 60 seconds was 0.573 (Figure 1: Table1). To calculate the concentration (molarity), the Beer’s Law equation was used, Abs = slope(m)+b. Plugging in what is known into the Beer’s Law equation resulted in 0.573 = 3.172e+004 + 0, where the concentration is determined by M = 0.573-0/ 3.172e+004. So, the concentration at 60 seconds using the equation (M = 0.573-0 / 3.172e+004) was 1.824e-5 M. The 1st order graph resulted in k=0.006152 (Figure 1: Graph 1). Other groups also resulted in their decolorization of CV to be the 1st rate

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.

The Beers Law calibration experiment used many concentrations of crystal violet solutions. Each of these solutions were test and analyzed in order to determine the absorbance of each concentration The results were than graphed and produced a slope of 1.00E05 with an intercept of -2.21E-02.

Make sure to use the same type of cuvette to keep the width consistent and to prevent any experimental error from arising. Obtain 5 of the same type of cuvettes and pre-rinse them thoroughly. Label them numbers one through five in increasing molarity. Then, fill each of the cuvettes with one of the five solutions you created back in Part A. We will first examine the solution that exhibits the highest concentration or molarity. Make sure to wipe the outside of the cuvette with a Kimwipe before placing into the SpectroVis Plus device. Observe the graph that is generated and make sure to take note where the maximum absorbance takes place.

The values of color absorbance are effective because color absorbance has a linear relationship with concentration values, which in turn, allows us to easily find concentration values for many solutions. Beer’s law describes this phenomenon since the absorbance is directly proportional to concentration. We observed that as the color absorbance increased, the concentration of the FeSCN2+ complex ion increased. This is because as the FeSCN2+ concentration increases, the blood-red color becomes darker due to more presence of the blood-red FeSCN2+ ion. Therefore, the color absorbance increases because there is more blue color absorbed by the darker red color. We then graphed the absorbance and concentration values and created a line of best fit. Using the line of best fit, we were able to predict the equilibrium concentrations of the FeSCN2+ solutions and find the change required to reach equilibrium. Since we already knew the initial concentration of FeSCN2+ and since we already found the equilibrium concentration of FeSCN2+, we can calculate the change in equilibrium. Using this data, we were able to calculate the equilibrium concentration of all of the species in this lab, since we already knew the change from the initial concentration to the equilibrium change. Q is less than K because there was no initial concentration of FeSCN2+, but after the system reached

b. Describe the spectral curve of each target type with respect to its absorption and reflectance characteristics. (3)

A = Absorbance difference  = Molar extinction coefficient C = Concentration L = Path length

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.

A spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. This instrument is usually used for the measurement of reflectance of solutions. Light is separate into different wavelengths and is being passed through the sample solution. The sample solution will have its own wavelength and will absorb a certain amount of light. The higher the molecular concentration, the higher the absorbance value.

concentration, record the absorbance readings at a fixed wavelength, and plot the absorbance vs. concentration data. The wavelength of 520 nm was selected for experiment Part