Spectrophotometry

SPECTROPHOTOMETRY - DETERMINATION OF PHOSPHOROUS

Spectrophotometry is  based on the Lambert – Beer’s law. It can be stated that the rate of decrease in the intensity of radiation with the thickness of the absorbing solution is proportional not only to the intensity of the incident radiation but also to the concentration of the solution.The variations in the intensity of the colour of a system due to the concentration of some component forms the basis of Spectrophotometry.             

When light (monochromatic or heterogeneous) falls on a homogeneous medium, a portion of the incident light is reflected, another portion is absorbed within the medium and remainder is transmitted.  This could be mathematically expressed as

I0 = Ia + It + Ir

Where    

                 I0 =Intensity of incident light

            Ia =Intensity of light absorbed

            It = Intensity of light transmitted

            Ir = Intensity of reflected light

Spectrometry measures the relative amount of light transmitted by an adsorbing medium. Spectrometric methods are more accurate for determining the concentration of the substances in solution than colorimetric techniques. In the spectrometric methods, the following regions of the spectrum are used. UV -180 to 400 mμ; Visible- 400 to 760 mμ or 760 mμ to 25μ. In the spectrometric analysis, light of wavelength not exceeding 1 to 10 A° in band width extending to the UV region of the spectrum is used as a source of light.

A spectrometer is made up of two instruments – a spectrometer and a photometer. A spectrometer produces coloured light of any selected colour or wavelength. A photometer measures the intensity of light and in a spectrophotometer it is used to measure the intensity of the monochromatic beam produced by the associated monochromator. Generally photometric measurements are made first with a reference liquid and then with a coloured sample kept in a similar cell interposed in the light beam, the ratio of the two intensity measurements being a measure of the capacity of the same at the testing.

S1: Disperse the light

S2: Select the desired wavelength

Colorimeters cannot be used to estimate concentrations of < 0.001 μg as there will not be any visible colour development.  But with a spectrometer even such low concentrations can be estimated. Also the methods are rapid and accurate and hence hundreds of samples could be analysed daily. Usually this instrument is used for analyzing samples containing 0.01 to 0.001 M concentration of the elements.

The essential parts of a spectrophotometer are a source of radiant energy, a monochromator, glass or silica gel for the solvent and for the solution under test and a device for receiving or measuring the beam or beams of radiant energy passing through the solvent or solution. The versatile nature of this instrument is due to the availability of wide range of wavelengths. A tungsten filament or incandescent bulb provides light for the measurement in the visible region of the spectrum, and a hydrogen gas discharge tube for the UV range. Glowing rods of silicon carbide or zirconium oxides are used in the IR region. Glass is not transparent to UV rays. Hence prism and windows are fabricated from inorganic halogen salts such as NaCl or KBr.       

Correction is always done by setting the instrument to zero absorbance using a blank. The present models have both transmittance and absorbance scales.

Procedure

P extraction by Olsen method

Take 2 g of the 2 mm sieved soil and transfer it to a 250 ml shaking bottle.  Add 40 ml of the Olsen reagent and a pinch of Darco G 60 (To make the solution colourless).  Shake in a horizontal shaker for 30 minutes.  Filter the contents through Whatman No.1 filter paper collecting the filtrate in a suitable container.  Estimate the P concentration in this extract by the ascorbic acid method.

Colorimetric estimation of P by ascorbic acid method (Murphy and Riley, 1962)

Reagents

(i)   Ammonium molybdate-Antimony potassium tartarate solution (Stock solution)

Dissolve 12 g of ammonium molybdate in 200 ml of warm water and cool.  Dissolve 0.291 g of antimony potassium tartarate in100 ml distilled water.  Add both the solutions to 1000 ml of 5 N H2SO4 (138 ml of con. H2SO4/l water), mix homogeneously and make-up to 2 litres with distilled water.  Store in a pyrex glass bottle in a dark and cool compartment.  This solution is heat and light sensitive (Reagent A).

(ii) Ascorbic acid reagent

Dissolve 1.056 g of ascorbic acid in 200 ml of reagent A. This reagent should be prepared freshly as and when required (Reagent B).

Procedure

Pipette out 5 ml aliquot of the soil extract of either Olsen or Bray method into a 25 ml volumetric flask.  Adjust the pH to 5. This can be done by taking 5 ml of the Olsen or Bray No.1 soil extract and assessing the volume 1 N H2SO4 or 1 N NaOH required to adjust the pH to 5.  Aqueous 0.25% solution of p-nitrophenol can be used as indicator which will be yellow at pH 5 and above and colourless at pH below 5.

After having adjusted the pH to 5, add 4 ml of the ascorbic acid reagent (reagent B), makeup the volume, and mix well and allow 20 minutes.  Measure the intensity of blue colour in a photoelectric colorimeter at 660 nm (red filter).  Prepare a standard curve with standard P solutions.  Find out the concentration of P in the soil sample extract using the standard curve and calculate the available P status in the soil from the P concentration in the soil extract.

Preparation of standard curve

Standard P solution

Prepare 1000 ppm P solution by dissolving 0.4390 g of AR grade KH2PO4 in distilled water and make up to 100 ml.  Dilute 10 ml of this solution to 100 ml with distilled water to prepare 100 ppm P solution.  Dilute 10 ml of the 100 ppm solution to 100 ml to prepare 10 ppm P solution.  Dilute 10 ml of 10 ppm P solution to 100 ml to prepare 1 ppm P solution.

 Pipette out required volumes of the standard P solutions into 25 ml volumetric flasks as given below and add 5 ml of the respective reagent (Olsen or Bray o.1 reagent). Adjust the pH to 5 using paranitrophenol indicator and 1 N H2SO4 or 1 N NaOH as the case may be.  Dilute to about 20 ml with distilled water.  Add 4 ml of the ascorbic acid reagent (reagent B), make up the volume to the mark and allow 20 minutes for colour development.  Measure the intensity of the blue colour in a photoelectric colorimeter at 660 mm (red filter) and draw the standard curve with the P concentrations in the X axis and the % T or O.D. in the Y axis.

P in ppm                                                  Volume of standard solution                               Volume to be made up

0.1                                                                 2.5 ml 1 ppm solution                                                   25

0.2                                                                 5.0 ml 1 ppm solution                                                   25

0.3                                                                 7.5 ml 1 ppm solution                                                   25

0.4                                                                 10.0 ml 1 ppm solution                                                 25

0.5                                                                 12.5 ml 1 ppm solution                                                 25

0.6                                                                 15.0 ml 1 ppm solution                                                 25

0.7                                                                 1.75 ml 10 ppm solution                                               25

0.8                                                                 2.0 ml 10 ppm solution                                                25

0.9                                                                2.25 ml 10 ppm solution                                               25

0.10                                                               2.5 ml 10 ppm solution                                                 25

Calculation

Weight of soil used for extraction= 2 g

Volume of extractant used = 40 ml

Volume of the extracted solution taken

 for P estimation = 5 ml

Final volume  made upto = 25 ml

Concentration of P read in the standard curve corresponding to the percent  transmittance = a ppm

Therefore, amount of available P in the soil =  a x 25/1000000  x 40/5 x 1000000  kg/h

Rating of soil with reference to available P status

Low :  Upto 11 kg ha-1

Medium :  More than 11 and upto 22  kg ha-1

High :  More than 22 kg ha-1