Description

LAB 4.          SPECTROPHOTOMETRIC ANALYSIS OF PHOSPHORUS

Introduction

Soil is a complex and variable mixture of rock fragments, organic matter, moisture, gases, and living organisms that provides mechanical support for growing plants. Soil also stores supplies of water and other critical nutrients for growing plants and as a result the productivity of the site is directly related to the productivity of the soil. The elements nitrogen, phosphorus, potassium and calcium are all essential to the growth and the development of both plants and animals.

Soil test phosphorus (STP) is not an indication of total P in the soil but how much is available for plant use. If STP numbers are to be compared, the laboratory test method for extracting P and how the number is reported (parts per million or pounds per acre) must be known. Different testing labs use different methods for extracting P, producing different test results that are difficult to compare even for the same sample.

Soil testing facilities may report results in pounds per acre of elemental P. Other soil testing labs may report their results in parts per million (ppm) without making the conversion to pounds per acre (lbs/A). This conversion from ppm to lbs/A involves assuming that a 6-inch deep layer of soil (furrow slice) covering one acre weighs 2,000,000 pounds. To convert soil test results from ppm to lbs/A, you’ll need to multiply the value in ppm by 2. For example, a soil test P value of 150 ppm is correlated to 300 lbs/A. In addition Kg P/ha = ppm X 10

About 99% of the total phosphorus in soils is tied up in compounds that are not readily available to plants. Approximately two-thirds of this total is inorganic-P. The remaining portion is organic-P. Phosphorus can be released into plant available forms from either of these sources. Inorganic-P often reacts with soluble aluminum, iron, or calcium in the soil solution to form relatively insoluble compounds. These reactions depend on soil pH. Inorganic-P can be relatively unavailable at either a very low or very high soil pH. Organic-P compounds are continually being immobilized and released by soil microorganisms and growing plants.

Of the three primary nutrients, phosphorus is the least mobile in the soil, so leaching of P into groundwater is not often a concern. This does not mean that phosphorus will not move off-site, however. Phosphorus, both inorganic and organic, can readily be transported to surface water along with eroding soil particles. Phosphorus can also be lost while dissolved in water.

Purpose

To introduce the student to spectrophotometric analysis of soil phosphorus through the preparation of standards and the determination of a calibration curve using a Bauch and Lomb spectrophotometer.

Prepared Reagents:

1.       1000 ml of Stock Phosphate solution (25 mg/l)

2.       Ammonium Molybdate Reagent

3.       Dilute Stannous Chloride Solution (Prepared daily)

Apparatus:

1.       1- digital pipette

2.       1-25ml volumetric flasks

3.       1-4ml volumetric pipette

4.       1-25ml burette

5.       1- test tube stand

6.       11-test tubes

7.       6- 50ml beakers

8.       1- spectrophotometer cuvet

9.       parafilm

10.     labels

Procedure:

1.       Determine the amount of Stock Phosphate Solution required to prepare 5 – 25ml standards of 1, 5, 10, 15, 20 mg/l using the following dilution formula:

C1V1 = C2V2

          Where          C1 = concentration of stock phosphate solution (ie. 25 mg/l)

                              V1 = volume of stock solution required to make a given standard

                              C2 = concentration of standard to be prepared

                              V2 = volume of standard to be prepared (ie 25 ml)

Table 1:  Amount of stock phosphate solution (25 mg/L) required to prepare standards of varying concentration.

2.       Preparation of Standards:

          Prepare standards by starting with the lowest concentration first (1 mg/l); using a

          25 ml burette, transfer the required amount of stock solution to a 25 ml volumetric     flask and dilute with distilled water to the 25 ml mark. Pour into an appropriately           labelled clean beaker.

3.       Set up 11 test tubes in a test tube rack.

          Using a 4-ml volumetric pipette, transfer:

·       4 ml of each of the standards (from Step #2) to a separate test tube and label each test tube (eg. 1, 5, 10, 15, 20 mg/l)

·       4 ml of the known sample (prepared samples) to a separate test tube. Repeat. Label each test tube (eg. Known trial #1, Known trial #2)

·       4 ml of the unknown sample (prepared samples) to a separate test tube. Repeat twice. Label each test tube (eg. Unknown trial #1, Unknown trial #2, Unknown trial #3)

·       4 ml of distilled water to a separate test tube and label as “Blank”

4.       Colour Complexing of the Standards, Samples and Blank

For all 5 standards, the one known (2 trials) and the one unknown (3 trials) and the blank (1)         proceed with the following procedure:

·       Using a digital pipette, add 1 ml of the molybdate reagent

·       Using a digital pipette, add 1 ml of the dilute stannous chloride solution

          Wrap the top of each test tube with parafilm and invert several times to mix.

          A blue colour should be present. Allow samples to stand for 10 minutes in a test tube rack and measure transmittance at 680 mm (red filter)

5.       Transfer the “Blank” to a cuvette and calibrate the meter to 100% (transmittance).

Pour your blank back into the test tube in case you need to calibrate the meter again.

6.       Starting with the lowest concentration, transfer the solution to the same cuvet,           wipe the outside of the cuvette, place in the spectrophotometer and analyze.        Record Absorbance. Use this same cuvette for all readings for the remaining 5   standards and record.

7.       Use the same cuvette as in #6 and the same methodology to analyze the known           concentration (2 trials) to validate the calibration curve, record

8.       Use the same cuvette as in #6 and the same methodology to analyze the           unknown concentration (3 trials), record