5. How Can You Determine the Best Wavelength to Analyze the Phosphate Ion Content of Solutions?

EXPERIMENT 36 - COLOURIMETRIC Decision OF PHOSPHATE

Goal: To introduce students to the employ of UV/Vis spectroscopy in belittling chemistry.

Objectives:

On completion of this lab you should exist able to:

1. Place the components of a UV/Vis spectrophotometer and the functions of each component.
2. Preparing a suitable set of calibration standards.
3. Mensurate absorbance of solutions at the specified wavelength.
4. Apply the Beer-Lambert police force and absorbance measurements to decide concentration of a coloured species in solution.
5. Construct and use scale curves to make up one's mind the concentration of an analyte in the analytical solution.

Introduction

In that location is oftentimes a direct human relationship betwixt the intensity of the colour of a solution and the concentration of the coloured component (the analyte species) which it contains. This straight human relationship forms the basis of the colourimetric technique. One might readily make up one's mind the concentration of a sample based on its colour intensity, simply by comparing its color with those of a serial of solutions of known concentration of the analyte species. In some cases the colour of the solution may be due to an inherent property of the analyte itself, for case, a KMnO₄ solution has a natural purple colour, the intensity of which can exist readily measured. In many other cases, yet, the solution color is developed by the addition of a suitable reagent which interacts with the analyte species thereby forming a coloured complex.

Colourimetric conclusion of phosphate

Theory

The amount of electromagnetic radiation in the visible region of the spectrum captivated by a coloured solution is often straight proportional to the concentration of the coloured species every bit divers past the Beer-Lambert Law, A = εcl.
Intensity of coloured solutions are normally measured with a spectrophotometer. A beam of calorie-free of intensity Io is focused on a sample, and a portion, I, is absorbed by the analyte species. The amount of calorie-free absorbed may exist mathematically expressed as:

A = log (I_o/I)       (1)

The absorbance, A, is related to concentration past the Beer-Lambert constabulary:

A = εcl                 (ii)

which states that the absorbance of a solution is directly proportional to its concentration, c, every bit long as the solution path length, l, and the wavelength of measurement are constant. Once the Beer-Lambert constabulary is obeyed, a plot of absorbance confronting concentration will give a direct line, the slope of which is the molar absorptivity, ε * length.

Colourimetric techniques are useful in the assay of a wide range of substances. Ane of import application is its utilise in determining the phosphate content of natural and wastewater sources. Phosphate is considered to be ane the virtually important nutrients in natural water. Although several other nutrients (eg. carbon, nitrogen, sulfur, potassium, calcium and magnesium) are required to facilitate growth of institute material, especially algae, the phosphorus content is critical in determining the level of algal growth that the water will support. The growth of algae in natural water will rarely occur at phosphate concentrations beneath 0.05 mg/dmⁱⁱⁱ. Drinking h2o may accept a maximum allowable phosphate content of 0.3 mg/dm3, while on boilerplate, raw sewage contains about thirty mg/dm³.

The phosphate found in natural waters mainly exists equally the orthophosphate species, PO₄ ^3-, however, the polyphosphates P_iiO₇ ^4- and P₃O₁₀ ^5- are frequently encountered. These polyphosphate species may be hydrolysed to produce the orthophosphate, withal, the species which dominates will depend on the pH prevailing in the detail surround.

Phosphate will readily react with ammonium molybdate in the presence of suitable reducing agents to grade a blue coloured complex, the intensity of which is straight proportional to the concentration of phosphate in the solution. The phosphate content of an unknown water sample can be obtained by first plotting the absorbances of a serial of standard solutions against the respective concentrations, thus giving a scale curve. The concentration of phosphate in the unknown sample can and so exist determined from the graph.

In this exercise, a sample of natural water has been provided which has been filtered and treated to remove all materials likely to cause interference. You are required to decide the phosphate content of the sample in indistinguishable, using the spectrophotometric technique outlined below.

Procedure

A. Grooming of Calibration Bend

Set up a standard stock solution of phosphorus of approximately 100 mg P/dm³ by dissolving 0.11g of KH₂PO₄ (this should exist accurately weighed) in distilled water and diluting to 250 cm³ in a volumetric flask (Stock Solution A).
Accurately transfer x cm^three of this solution to a 250 cm^three volumetric flask (Stock solution B) and brand upwards to volume with distilled water.
Use stock solution B to ready standards of approximately 0.20, 0.40, 0.60, 0.eighty and i.0 mg P/dm³, that is, pipette 5, 10, 15, 20 and 25 cm^three portions respectively to separate labeled 100 cm³ volumetric flasks. Identify roughly 50 cm³ of distilled water into a 100 cm³ flask as a blank solution, then organize all the belittling solutions for colour development.

Do non make up the solutions to the mark yet.

B. Analysis of Water Sample

You are provided with a water sample that has been diluted by a gene of 10. Pipette indistinguishable 25.0 cmⁱⁱⁱ portions of the diluted sample to two separate 100 cm³ volumetric flask, then develop the colour as outlined below.

C. Colour Evolution

Add distilled water to all the analytical solutions (standards and samples) so that each flask contains roughly l cm³ of solution. Starting with standard 1, add thirteen cmⁱⁱⁱ of combined reagent using a 25 cm^three measuring cylinder (Note one). Shake thoroughly and brand up to the marking with distilled h2o. Treat all the solutions similarly and so allow 30 minutes for colour evolution.

Prior to measurement, prepare the wavelength of the instrument to 880 nm then null with distilled water in a i-cm cuvette. Measure the absorbances of the standards in society of increasing concentration followed past that of the sample solutions.

Tape your results in the table provided then plot a graph of the corrected absorbance vs the corresponding concentration. Consummate the exercise past answering the questions and performing the calculations on the laboratory worksheet.

General Notes

ane. Combined Reagent: Prepared by combining 500 cm³ of ii.5 M H₂So₄, 50 cm^three potassium antimony tartrate solution (i.e. prepared by dissolving 1.371 g K(SbO)C₄H₄O_six.0.v H₂O in well-nigh 400 cm³ distilled h2o and diluting to 500 cm³) and 150 cm³ ammonium molybdate solution (20 g (NH₄)_half-dozenMo₇O₆.4H₂O in 500 cm³ of water), 300 cmⁱⁱⁱ ascorbic acid solution (made by dissolving 5.28 g of ascorbic acrid in 300 cm^three of H₂O). The solution is thoroughly shaken and stored in plastic bottles. A fresh mixture is made on the morning of each lab solar day.

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