Quantitative Analysis by Spectrophotometric Methods

The concentrations and absorbances of the red and blue dyes were used to find the concentration of the purple dyes. From the graph of the blue dye, the linear equation for absorbance was y = mx + b. From that formula came the equation y = 7.915 x 104 (x) + 0.02489, where y represents absorbance, m is slope, x is concentration/molarity, and b is the constant/y-intercept. The same set up was performed for the red dye, but the equation produced was y = 1.045 x 104 (x) +.001298. The equations found when graphing absorbance vs. concentration were used to find the concentration of the purple dyes. The absorbance for purple dye 3 on the red wavelength of 470 nm equaled 0.149 and 0.818 for the blue wavelength of 635 nm. For purple dye 1

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.

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

The ending result of this experiment confirms that as five test tubes are lined up with the varying level of absorbance, different results in the level of absorbance will appear as well, this is visible in above table. Thus, this is due to the varying amount of water in the solution. The blank sample had a 0.30 in its level of absorbance.

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.

Organisms that use the process of photosynthesis to create sugar to use for energy have a greater rate of photosynthesis when the intensity of the light source is the greatest. If light is far away from the leaves of a plant, for example, then it takes more time for the light to be absorbed and used in photosynthesis. When it takes more time for the light to reach the leaves, the rate of photosynthesis decreases. As the light intensity increases, I would expect the rate of photosynthesis to increase as well. Therefore, I would expect that when the Elodea is closest to the light bulb, the rate of photosynthesis would be the greatest. My hypothesis would be: If light intensity affects the rate of photosynthesis, and the rate of photosynthesis is measured using the amount of

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.

MANUAL Q5: What color of light is being absorbed by the sample solution? How is that color related to the color of the solution?

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

Where A is the initial absorbance when the experiment first starts, l is the path length of the cuvette (2.54 cm), and [CV]t is the initial concentration of crystal violet.

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

Figure 2: Dye percents versus absorbance in a control, 10%, 20%, and 30% azide solutions.

3. The spectrophotometer was set at 420nm. Distilled water was also used as the ‘blank’.

We have timed each of the three experiments for 5 minutes to make sure that our results are correct. Thanks to the program, we got the transmittance and the absorbance, together with the graph portraying our results.