General chemistry laboratory
Fall 2014
Assay for total phosphates
Phosphorous is an essential plant nutrient, and is present in soil in both organic
(e.g., as part of DNA, ATP) and inorganic (e.g., PO4-3, H2PO4-) forms.
Soil composition
Soil is composed of minerals, organic material, air, and water. The mineral in
soils comes from small bits of weathered minerals – weathered rock. Silicate minerals are
the most common, although aluminum is also a common component. They are both
bound to oxygen atoms, although some combinations of Si4+, Al3+ and O2- also contain
Na+, K+, Mg2+, Ca2+, Fe2+, e.g., feldspar: KAlSi3O8, NaAlSi3O8, CaAl2Si2O8. After lots of
weathering and chemical reactions the particles can be small and somewhat varied in
composition. Really small particles – less than 2 m – are considered to be part of the
clay fraction of soil.
Organic material gives soil its dark color and is called humus. Humus is
comprised of decomposed plants, insects, bacteria, and fungi.
Soil nutrients
Plants need many nutrients to grow and reproduce, but those required in the
largest quantities are nitrogen (N), phosphorous (P), and potassium (K). Bags of fertilizer
you buy at the garden center list the percentages of these macronutrients as N:P:K. So a
bag of 10:10:10 contains 10% of each, and the remaining 70% is filler of little or no
nutritional value to the plant.
On Marlboro’s farm we use compost to provide these nutrients, rather than storebought fertilizer.
The Beer-Lambert law
Many applications of the spectrophotometer are made possible by a relationship
described in the Beer-Lambert law. For example, the colorimetric assay you will use for
measuring phosphorous is based on this relationship. To use the law we employ a
machine that directs a beam of light of know wavelength at an aqueous sample. A
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detector on the other side of the sample measures how much light has passed through the
sample.
The Beer-Lambert law relates the absorbance of a particular wavelength of light,
by the sample, to the concentration of some compound in solution:
Absorbance =  x concentration x path length, or A=•c•l
“Path length” is the linear distance the beam of light travels through the sample.
 is a proportionality constant, and is specific for a particular compound. It is known as
the “molar absorption coefficient” – the amount of light absorbed by a 1 M solution of a
particular compound, in a particular solvent, at a particular wavelength, with a light-path
(aka pathlength) of 1 cm. Therefore,  has units of M-1cm-1
Assay for total phosphates
You are going to use a spectrophotometer to quantify the amount of phosphorous
in your samples. To do this you have to construct a ‘standard curve’: You will make a
series of iron phosphorous solutions (aka ‘standards’), each containing a known amount
of P. You add specific reagents to each of these solutions; the chemical reaction between
P and the other reagents produces a product that has a blue-ish color. You will use the
spectrophotometer to measure the amount of blue color in each solution, then construct a
graph of “amount of blue color” vs. the known iron concentration of each ‘standard.’
Connecting the points on this line will give you a straight line – this is your ‘calibration
line’ or ‘standard curve.’
You will then compare the “amount of color” in samples from soil and compost
with that ‘standard curve’ to see how much P is in those samples.
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Extracting P from soil with water:
Weigh out 2 g of soil (sieved through a 2-mm mesh sieve) into a 40 mL centrifuge tube.
Add 20 mL of distilled water and shake for 5 min.
Centrifuge at 6,000 rpm for 10 minutes.
Filter the solution through two layers of Whatman No. 1 filter.
Reagents
Stock phosphate solution (50 mg P / l)
 219.5 mg potassium dihydrogen phosphate (KH2PO4) in 1000 ml deionized water
Standard solution (2.5 mg P / l)
 50 ml of stock phosphate solution up to 1,000 ml with de-ionized water
Standard curve
 construct a series of 8 standards from about 20 to 200 µg P/l
 be sure to include a blank in your set of standards
Reagents
A. 95% ethanol
B. 5N sulfuric acid
 70 ml conc. Sulfuric acid to 500 ml of de-ionized water
C. Antimony potassium tartrate
 4.39 g K(SbO)C4H4O6•1/2H2O in 200 ml de-ionized water
D. Ammonium molybdate
 20 g (NH4)6Mo7O24•4H2O in 500 ml de-ionized water
E. 0.1 M Ascorbic Acid
 1.76 g ascorbic acid in 100 ml de-ionized water
Combined reagent
 To make 100 ml of combined reagent, mix:
o 50 ml of B
o 5 ml C
o 15 ml D
o 30 ml of E
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Assay procedure for standards and samples

Mix the following components together in one well of a microplate:
 200 µl sample or standard
 10 µl 95% ethanol
 10 µl combined reagent


mix thoroughly and let stand for 10 min
measure absorbance at 710 nm
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