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How to use a spectrophotometer and understand how it works. -Understand the correlation and usage of Beers law with calibration curves. Introduction/ Procedure: Environmental protection has been a growing concern for many people in the recent years. With talks Of global warming, deforestation, and rising ocean acidity in the news it is safe to assume that protecting our environment is a task everyone will need to be a part of.

One example of environmental harm caused by humans is the leaking of phosphates into water supplies through the use of garden fertilizers. When there is too much phosphate in the water it causes process of triplication. Triplication is where the level of algae and marine life plant matter bloom to disastrous levels[3]. With the extreme amounts of algae in the water the oxygen supply of the water is diminished and marine life is destroyed[3]. While very small amounts of phosphates are necessary for the growth of roots in plant life the levels of phosphate added to water from fertilizers are dangerous for the ecosystem[2].

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In order to lactate the concentration of phosphate in water the molar absorptive must be found first. For this experiment using a stock phosphate solution is ideal as the concentration (in pimp) of phosphate can be controlled. First, six samples of varying phosphate concentration (0. 5, 1. 0, 2. 0, 3. 0, 4. 0, 5. 0 pimp) will be made by diluting 20 pimp stock phosphate solution with denizen water. This can be achieved by using the dilution equation: MI VI-?MOVE. Due to phosphate being uncontrolled an ammonium moldboard will be used to react with phosphate in the water to form a phosphorescently complex.

This employ will change color when reacted with a solution turning it a dark blue color. When the complex has changed color it can be placed into 1 CM civets and into a Spectrophotometer 200. A spectrophotometer works by measuring the intensity of light before and after entering the complex which allows the spectrophotometer to determine the absorptive at that wavelength. With the absorbent of each dilution found by using a spectrophotometer a calibration curve can be made.

With absorbent on the y-axis and the concentration on the x-axis of the curve the slope-intercept form can be found using excel. Slope-intercept form is equal to Beer’s law which will be needed to find the molar absorptive Of phosphate. Beer’s law is as follows: [2] Beers law can be correlated with slope intercept formula of y=mix+b where y = A, m=el, C=x, and b y-intercept]. Once the molar absorptive of the phosphate solution is found then the concentration of phosphate could be found in normal tap water or unknown samples.

Results: In order to create the dilutions a calculated amount of denizen water had to be added to each solution containing the 20 pimp stock phosphate solution using MI FL=MOVE. Example: (0. (20 pimp)(VI) Table 1: Dilution Calculations Phosphate dilution desired (pimp) Denizen Water added (ml) 0. 5 pimp 2. 5 ml 1. 0 pimp 2. 0 pimp 10 ml 3. 0 pimp 15 ml 4. 0 pimp 20 ml 5. 00 pimp 25 ml Once the dilutions were made the absorbent was found using a spectrophotometer: Table 2: Absorbent of Solutions Phosphate Concentration (pimp) Absorbent 0. 16 o . 40 0. 50 0. 70 1 . 00 5. 0 pimp 1. 8 Once the absorbent was found a calibration curve can be made using the phosphate concentration on the x-axis and absorbent on the y-axis. Graph 1 : Absorbent of Phosphate Concentrations at 650 NM. Once the trend-line (y = 0. 2171 x + 0. 0958) was obtained the concentration of phosphate can be calculated in the unknown by using the absorbent of the unknown sample. Table 3: Absorbent of Unknown A Trial # Trial 1 0. 25 Trial 2 0. 26 Trial 3 The concentration of phosphate in the unknown sample could now be found using the trend line equation from the stock phosphate calibration curve.

Example: 0. 25=0. Xx+0. 0958 Table 4: Phosphate concentration in Unknown A Trial / Absorbent Concentration Trial 1 / 0. 25 0. 710 pimp Trial 2/0. 26 0. 756 pimp Trial 3 / 0. 25 Average: 0. 725 pimp standard Deviation: 0. 2656 The stock phosphate solutions were all diluted to the correct amount using the calculations from table 1 . Then each dilution was put into the spectrophotometer finding the absorbency’s of each dilution shown in table 2. Using the absorbent and concentrations a trend line was created on graph 1 to find the molar absorptive of phosphate.

Meanwhile, the absorbent of Unknown A was found once again by using a spectrophotometer in table 3. Using the trend line from graph 1 and the absorbent from table 3 the concentration of phosphate in the unknown was calculated in table 4. Discussion: The correlation between increase in absorbent and the increase of phosphate in the water seemed accurate as the absorbent of each dilution should have increased as the amount of phosphate increased. Overall, the absorbent dilutions were fairly precise as the RAY value was very close to one (perfect precision).

It was observed that as the dilutions increased the color of each solution would turn a darker blue. This was expected as the darker blue correlated with increase of phosphates in each dilution. The absorbent of the unknown were also very precise with only trial 2 differing from the other trials. It was expected that all the trials of the unknown should have had the same absorbent. It is likely there was either a smudge on the cavetti or an error made with the preparation of the unknown in trial 2 resulting in a different absorbent.

The concentrations of the unknown seemed accurate as the concentration recorded lied within their respective boundaries found through the dilution data. In other words: At 0. 25 absorbent the unknown concentration should have been between 0. 5 and 1. 0 pimp using the data from the stock phosphate dilutions. The standard deviation showed that the data was not as precise as desired due to trial 2 Ewing off in absorbent. Some sources of error throughout the lab could include not properly cleaning the flasks after every trial which could make the concentrations increase in the experiment.

Overall, the data seemed to be great with high precision and more than likely decent accuracy based off of assumptions. Conclusion: The objectives presented in the beginning of the experiment were easily accomplished throughout the lab. The concentration Of phosphate in the unknown is high and if obtained from a local water source should probably be investigated. Normal amounts of phosphate in water should be under 0. 1 pm, anything over can result in triplication [11.

It would not be unlikely to find high levels of phosphate in Indiana waters as the state relies heavily on agriculture and fertilizer.

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