Interdisciplinary connections

• Chemistry, Biology, Environmental Science, Social Studies, English

Teacher Notes

Principle

• Copper II  ions form a water-soluble orange-colored chelate with bathocuproine disulfonate (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid, disodium salt).  While the color forms over the pH range 3.5 to 11.0, the recommended pH range is between 4 and 5.  The sample is buffered at a pH of about 4.3 and reduced with hydroxylamine hydrochloride.  The absorbance is measured at 484 nm.  The method can be applied to copper concentrations up to at least 5 mg/L with a sensitivity of 20 mg/L.

Apparatus suggestions

• Colorimetric equipment with a light path of 1 to 5 cm (unless nessler tubes are used) might be either a spectrophotometer for use at 484 nm, filter photometer equipped with a blue-green filter exhibiting maximum light transmission near 484 nm, or a Calculator-Based Laboratory (CBL) system which uses a colorimeter probe. Acid-washed glassware: rinse all glassware with concentrated Hydrochloric acid (HCl) and then with double distilled water.

Reagent preparation

• Double distilled water:  Prepare copper-free water for use in reagents and dilutions, by distilling singly distilled water in a resistant-glass still, or distilled water passed through an ion exchange unit.
• Stock copper solution:  To 20.0 mg polished electrolytic copper wire or foil in a 250 mL conical flask, add 10 mL water and 5 mL concentrated Nitric acid (HNO₃).  After the reaction has slowed, warm gently to complete dissolution of the copper and boil to expel oxides of nitrogen, using precautions to avoid loss of copper. Cool, add about 50 mL water, transfer quantitatively to a 1 L volumetric flask, and dilute to the mark with water; 1 mL = 20.0 mg copper.
• Standard Copper solution:  Dilute 250 mL stock copper solution to 1000 mL with water; 1.00 mL = 5.00 mg Cu. Prepare daily.
• Hydrochloric acid (HCl):   Prepare as a 1:1 ratio of acid to water.  WARNING: BE SURE TO ADD THE ACID INTO THE WATER.
• Hydroxylmine-hydrochloride (NH₂OH*HCl) solution:  Dissolve 50 g of NH₂OH*HCl in 450 mL of water.
• Sodium citrate (Na₃C₆H₅O₇*2H₂O) solution:  Dissolve 300 g Na₃C₆H₅O₇*2H₂O in water and make up to 1000 mL in a volumetric flask.
• Disodium bathocuproine disulfonate - C₁₂H₄N₂(CH₃)₂(C₆H₄)₂(SO₃Na)₂ solution:  Dissolve 1.00 g  C₁₂H₄N₂(CH₃)₂(C₆H₄)₂(SO₃Na)₂ in water and make up to 1000 mL in a volumetric flask.

Materials & Equipment Needs

Background

Search Words:

• Search Words: phytoremediation, heavy metals, rhizosphere, phytoextraction, bioremediation, rhizofiltration, phytostabilization, hyperaccumulators

Student Lab

Phytoremediation is the use of green plants to remove, contain, or render harmless an environmental contaminant.  Phytoextraction of heavy metals involves using metal-accumulating plants to transport and concentrate metals from the soil into harvestable parts of roots and above-ground shoots.  Rhizofiltration involves plant roots and their ability to absorb, precipitate and concentrate toxic metals from polluted sites. Heavy metal tolerant plants are also used to reduce the mobility of heavy metals, thereby reducing the risk of further environmental degradation by leaching into the ground water or by airborne spread.  Conventional cleanup techniques are not only destructive, but they are expensive.  Using terrestrial plants for metal accumulation and environmental remediation is certainly a cost-effective alternative to these approaches.

Hypothesis
 
 

Materials
 

Reagents	Materials	Apparatus
Distilled water	Acid-washed glassware	Spectrophotometer
Polished electrolytic copper wire or foil	250 mL round bottom flask	25 mL cuvettes
Concentrated nitric acid  (HNO3)	1 Liter volumetric flask	Analog or digital pH meter
Concentrated sulfuric acid (H2SO4)	Lettuce, mustard, radish or other seed selections
Hydroxylmine-hydrochloride (NH2OH*HCl)	Potting soil
Sodium citrate (Na3C6H5O7*2H2O)	Paper towels
Disodium bathocuproine disulfonate  C12H4N2(CH3)2(C6H4)2(SO3Na)2	Plastic disposable pipettes
	2 Liter plastic soda bottles
	250-mL Erlenmeyer flask

Chemical Protocol
 
To make reagents suitable for testing copper sulfate in plants, students may use the following protocol:
Teacher could prepare chemical solutions and students can develop their standard curve or students could prepare the chemical solutions and their own standard curves.  (See teacher notes)

Standard Curve Protocol

Pipet 50 mL sample, or suitable portion diluted to 50 mL, into a 250-mL Erlenmeyer flask.  In separate 250-mL Erlenmeyer flasks, prepare a 50 mL water blank and a series of 50 mL copper standards containg 5, 10, 15, 20, and 25 mg copper.  To sample, blank, and standards add ( mixing after each addition) 1 mL 1+ 1 HCL, 5 mL NH₂OH*HCl solution, 5 mL sodium citrate solution, and 5 mL disodium bathocuproine disulfonate solution.  Transfer to cuvettes and read sample absorbance against the blank at 484 nm.  Plot absorbance against micrograms Cu in standards for the calibration curve.  Estimate concentration from  the calibration curve.

Calculation

mg Cu/L = ^{mg Cu (in 66 mL final volume)
                                    mL sample}
See Student Lab section