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Spectrophotometric Determination of Phosphorous

Coral, Ralph Kristianne Duane Coral

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It deals with visible light, nearultraviolet, and near-infrared and involves the use of a spectrophotometer, which is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. In this experiment, the students were tasked to determine the absorbance and concentration of the sample that was placed in nine (9) flasks with different concentrations of the working phosphate solution. After placing the different concentrations of the sample in the spectrophotometer, the results were then recorded and analyzed. The concentration of the phosphate solution (Stock and Phosphate) were found to be 50.01 ppm P and 0.5 ppm P respectively. Different concentrations (in ppm P) of the flasks from 1 to 9 are 0, 0.005, 0.015, 0.025, 0.050, 0.100, 0.150, 0.250, and 0.450 respectively.

Phosphorous is the eleventh (11th) most abundant element on the surface of the earth and is commonly found in the form of phosphate (PO4

). It is a component of DNA, RNA,

ATP, and also the phospholipids that form all cell membranes. It is thus an essential element for all living cells, and organisms tend to accumulate and concentrate it. Spectrophotometry is study of the interaction of light with matter, and is an important and versatile tool for the chemist. Indeed, much of our knowledge of chemical substances comes from their specific absorption or emission of light. In this experiment, we are interested in the analytical procedures based on the amount of light absorbed (or transmitted) as it passes through a sample, and determining the concentration of phosphorus in it.

Spectrophotometric Determination of Phosphorus

A spectrophotometer was used by the students in determining the absorbance and of the sample tested. It consists of two instruments, a spectrometer which produces light of any selected wavelength or color, and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.

Experimental Section Chemicals and solutions All solutions were prepared with analytical grade reagents that were premade by the lab technicians and distilled water. The stock phosphate solution was made by dissolving a weighed amount of 0.2197 g of KH2PO4 in distilled water and quantitatively diluting it to 1000.0 mL in a volumetric flask. From the stock solution, a working solution was made by diluting 10.0 mL of the stock solution to 1000.0 mL. A coloring reagent (mixed reagent) was made by placing 50.0 mL of 5M sulfuric acid in a 250 mL beaker and by slowly adding and stirring (in order) 5 mL of K(SbO)C 4H4O solution, 15 mL (NH4Mo7 4H2O), and 30 mL ascorbic acid. Apparatus A double bean spectrophotometer was used in order to determine the absorbance of the sample. It is composed of a light source which emits a monochromatic light; a wavelength selector for picking out the desired wavelength; a chopper which cuts the light; a detector which tells how much light was absorbed; and a readout from the computer; and a pair of cuvette to hold the sample.

Spectrophotometric Determination of Phosphorus

The intensity of light passing through the blank is measured. The intensity refers to the number of photons per second. The blank is a solution that is identical to the sample solution except that the blank does not contain the solute that absorbs light. This measurement is necessary, because it will be the basis or reference of the analysis. Then the intensity of light passing through the sample solution is measured. Figure 1

Figure 1 [4] shows how a spectrophotometer works and displays its parts. Procedure Before anything was done, all the glasswares to be used were rinsed with 5M H2SO4 and distilled water to remove any trace phosphates from the soap/detergent that was previously used prior to the experiment. The Stock Phosphate solution was carefully prepared by the members of Group 6 by dissolving a weighed amount of 0.2197 g of KH2PO4 in distilled water and quantitatively diluting it to 1000.0 mL in a volumetric flask. Then a Working Solution was made by Group 7 by obtaining a volume of 10.0 mL of the stock solution and diluting it to 1000.0 mL.

Spectrophotometric Determination of Phosphorus

A coloring reagent was then prepared by a member of each group by placing 50.0 mL of 5M sulfuric acid in a 250 mL beaker and by slowly adding 5 mL of K(SbO)C 4H4O solution, 15 mL (NH4Mo7 4H2O), and 30 mL ascorbic acid while stirring in that order. Nine very clean 100.0 mL volumetric flasks were prepared and labeled 1-9. The volume of the components of the solution are as follows: Table 1 Amount of Solution, Sample, and Coloring reagent per flask
Flask number Working Phosphate solution (mL) Sample (mL) Coloring reagent (mL) 1 0.0 0 8 2 1.0 0 8 3 3.0 0 8 4 5.0 0 8 5 10.0 0 8 6 20.0 0 8 7 30.0 0 8 8 50.0 0 8 9 0.0 80 8

Table 1 [7] shows the volume of the different constituents of the solution for each flask. After placing the assigned volume on the different flasks, the solution (from flask 1-9) was then diluted to a volume of 100.0 mL. A double beam spectrophotometer was used in the analysis of the samples. It compares the light intensity between two light paths, one path passing through the blank sample and the other passing through the test sample. A sample from each flask was taken and placed one at a time on the spectrophotomer using a cuvette, which is a small rectangular container that is made up of quartz and consisted of a frosted side and a clear side. The cuvette must be handled with care because it is very fragile and very expensive. There were 2 cuvettes that must be filled with the solution, the first being the blank and the other for the samples. The blank was taken from Flask 1. In transferring the solution from the flask to the cuvette, one must wash the inside of the cuvette first with the sample to be tested and analyzed, then dry it with a lens paper without shaking the cuvette. The clear side must never be touched, only frosted side, on the fear that fingerprints might alter the reading of the sample. The blank cuvette must be placed all throughout the spectrophotometric process until finished. The solution from Flask 2 was then placed in another cuvette on the side nearest to

Spectrophotometric Determination of Phosphorus

the one conducting the experiment and let the machine analyze the sample; then Flask 3, and so on. After the experiment was done, all the glasswares were cleaned and dried.

Results and Discussion

Spectrophotometry is the study of the reflection or transmission properties of a substance as a function of wavelength. It is the quantitative study of electromagnetic spectra of a material. During the process, the transmittance or reflectance of the substance is measured through the careful geometrical and spectral consideration. Suppose you look at two solutions of the same substance, a deeper colored solution, and a lighter one. Your common sense tells you that the deeper colored one has the more concentration of the sample. In other words, as the color of the solution deepens, you infer that its concentration also increases. This is an underlying principle of spectrophotometry: the intensity of color is a measure of the amount of a material in solution. A second principle of spectrophotometry is that every substance absorbs or transmits certain wavelengths of radiant energy but not other wavelengths. Take the chlorophyll for example; it always absorbs red and violet light, while it transmits yellow, green, and blue wavelengths. The transmitted and reflected wavelengths appear green or yellow, which the color our eye sees. Therefore, the absorption or transmission of a specific wavelength is somewhat its characteristic or its fingerprint for a substance. The spectophotometric method for the determination of phosphorus is based on the ammonium molybdate reaction (which will turn the solution yellow on the first part of the reaction), followed by the reduction of the acidic medium (sulfuric acid) by the ascorbic acid, which is then catalyzed by antimony potassium tartrate which will hasten the reaction. The effect of adding sulfuric acid on absorbance was investigated to achieve high absorbance. It was proposed that the more acidic the medium, the more it made the solution sensitive; because phosphate is colorless, it does not absorb any visible wavelength, therefore, it was used throughout the experiment to determine the phosphate. Upon the addition of the reducing agent, ascorbic acid, the solution turned blue; the shade depended on 5

Spectrophotometric Determination of Phosphorus

the flask that it was placed. The intensity of the color blue is proportional to the amount of phosphate in the solution. The order of reagents addition to the solution did not affect the absorbance values. It was only done so as to maintain the uniformity of the order of reagents addition as mentioned in the recommended procedure was followed throughout course of the determination of phosphate. The reason why it was not washed with soap is that soap contains phosphorus which may alter the results and it may be included in the concentration of the solution. A stock solution was made but was not used directly in conducting the experiment, but the working solution was used. This is because the stock solution has a high concentration which will result to error. If the working solution, on the other hand, was made directly without the stock solution, it is very difficult to make a working solution because its concentration is not that high and the weight needed of phosphate is very low. The working solution is not stable and its concentration of phosphorus might be absorbed by the glass and can change the concentration over time. The purpose of the blank is for the spectrophotometer to know that the concentration of the blank is 0, and will serve as the reference solution all throughout the analysis. A double beamed spectrophotometer was used because there were two (2) cuvettes used in the analysis, one for the blank, and the other one for the test sample. The cuvettes were made of quartz because glass absorbs UV, so the wavelength will not pass through the sample; but, the students did not use UV light, but an ordinary light instead. The cuvettes were also rectangular in shape for the light to pass to equal path lengths and to have equal absorbance.

Figure 2



Figure 2 (a) shows the path equal path length of light when a square or rectangular container is used; (b) uses a circular container resulting to unequal path length of light.

Spectrophotometric Determination of Phosphorus

The wavelengths 650 nm and 880 nm were used because these are the two (2) highest or maximum wavelengths that is enough to be detected even in small concentrations of the solution. The blue color of the solution is because red and yellow are absorbed, while blue is transmitted not absorbed. Computation of the concentration of Stock Phosphate Solution (in terms of ppm P) ppm P = (weight in g KH2PO4) x (atomic weight of P) x (1000mg) (molecular weight P) 1L (1g)

ppm P = (0.2197 g KH2PO4) x

(30.97 g/mol P) (136.09 g/mol KH2PO4) 1L

x (1000mg) (1g)

ppm P = 50.01 ppm P

Computation of the concentration of Working Phosphate Solution (in terms of ppm P) C2 = (C1) (V1) V2 C2 = (50 ppm P) (10.0 mL) (1000 mL) C2 = 0.5 ppm P Computation of the concentration of Flasks 1-9 (in terms of ppm P) C2 = (C1) (V1) V2 Flask 1: C2 = (0.50 ppm P) (0.0 mL) (1000 mL) C2 = 0.00 ppm P Flask 2: C2 = (0.50 ppm P) (1.0 mL) (1000 mL) C2 = 0.005 ppm P 7

Spectrophotometric Determination of Phosphorus

Flask 7: Flask 3: C2 = (0.50 ppm P) (3.0 mL) (1000 mL) C2 = 0.015 ppm P Flask 8: Flask 4: C2 = (0.50 ppm P) (5.0 mL) (1000 mL) C2 = 0.025 ppm P C2 = (0.50 ppm P) (50.0 mL) (1000 mL) C2 = 0.250 ppm P C1 A1 Flask 5: C2 = (0.50 ppm P) (10.0 mL) (1000 mL) C2 = 0.050 ppm P C2 = C1 A2 A1 Flask 9: C2 880 nm= (0.25 ppm P) (1.115) Flask 6: C2 = (0.50 ppm P) (20.0 mL) (1000 mL) C2 = 0.050 ppm P (0.166) C2 = 1.68 ppm P C2 650 nm= (0.25 ppm P) (0.773) (0.104) C2 = 1.86 ppm P C2 = 1.86 + 1.68 C2 = 1.77 C2 A2 C2 = (0.50 ppm P) (30.0 mL) (1000 mL) C2 = 0.150 ppm P

Table 2 Absorbance and Concentration of Samples 1 to 9

Spectrophotometric Determination of Phosphorus

Sample 1 2 3 4 5 6 7 8 9

650 nm 0 0.002 0.007 0.013 0.001 0.026 0.035 0.104 0.773

880 nm 0 0.002 0.015 0.024 0.006 0.042 0.059 0.166 1.115

Concentration (in ppm P) 0 0.005 0.015 0.025 0.050 0.100 0.150 0.250 1.77

Table 2 [7] shows the absorbance of the sample (650 nm and 880 nm) and its concentration in ppm The results above were obtained by teams who followed the procedure correctly.

Spectrophotometric Determination of Phosphorus

Figure 3

Figure 3 shows the statistical graph of 650 nm (Absorbance vs. Concentration) which is based on Table 2. Figure 4

Absorbance vs. Concentration

1.4 1.2 1 Absorbance 0.8 0.6 0.4 0.2 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Series3 Series4 650 nm 880 nm


Spectrophotometric Determination of Phosphorus

Figure 4 shows the statistical graph of 880 nm (Absorbance vs. Concentration) which is based on Table 2. Two laws express the relationship between the absorption of light and the absorbing medium. According to Bouguers (or Lamberts) law, each layer of equal thickness of the medium absorbs an equal fraction of the energy traversing it. On the other hand, according to Beers law, the absorptive capacity of a dissolved substance is directly proportional to its concentration in a solution. If the shade or depth of color is associated with the concentration of a substance in a solution then that concentration can be measured by determining the extent of absorption of light at a specific wavelength. When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light. There are deviations to the Beer Lambert law. These are due to several factors [1]: Deviations in absorptivity coefficients at high concentrations (> 0.01M) due to electrostatic interactions. Scattering of light due to particulates in the sample. Fluoresecence or phosphorescence of the sample. Changes in refractive index at high analyte concentration. Non-monochromatic radiation

Conclusion The students found out that the concentration of a substance on a solution is linked with the shade of the color of the solution. They were also able to calculate the concentration of the solution and analyze the absorbance of it, with the help of a spectrophotometer. The students also concluded that if the concentration of a substance is increased, the color of the solution also increases with it. They also realized that the absorption or transmission of a specific wavelength is somewhat the characteristic or the fingerprint for a specific substance; the color of a particular material is dependent on the wavelength it absorbs or transmits. 11

Spectrophotometric Determination of Phosphorus

Acknowledgements The author would like to thank his team mates, Patricia Caindec, Jennifer Catapang, and Nicole dela Cruz, Sergie Fernandez, Eloisa Galvez, Dominic Garcia, and Christle Genesis; and also Ha Young Lee and Lorenzo Terania for helping and expressing their support in performing the experiment of Spectophotometry and suggesting different ways on how to do the paper. The author would also like to thank Tiffani Miranda for serving as an inspiration and for providing motivation, all throughout in the development of the research paper. The author would also like to express his gratitude to Prof. Rene Dilan and Asst. Prof. Christina Ramirez for guiding and helping the students in conducting the experiment and explaining the whys and hows of each detail.

References: [1] [2] [3] [4] [5] [6] [7] Laboratory Manual in Analytical Chemistry: For non-chemistry majors.