School Science Lessons
(UNChemLigand)
2025-06-27

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Ligands, EDTA
Contents
1.0 Ligands
1.3 Reactions of metals with ligands
2.0 EDTA
19.4.2 Electrophoresis, food dyes, marking pen ink, (Experiments)
2.5 Ion exchange resins, deionized water
Experiments
1.1 Copper (II) sulfate solution with ammonia solution, ligand substitution
1.2 Copper (II) sulfate solution with concentrated hydrochloric acid, ligand substitutionn
2.1 Mineral deficiency experiment, hydroponics, EDTA
2.2 Tests for metal ions in water, EDTA chelates
2.3 Tests for water hardness, EDTA titration, Calmagite indicator
2.4 Tests for water hardness, EDTA titration, Eriochrome Black T indicator

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1.0 Ligands
A ligand can be an ion or an atom that can form a coordination complex with a central atom, usually a metal.
A ligand is a molecule that selectively binds to another molecule.
Transition metals (transition elements) can form bonds called dative covalent bonds by accepting pairs of electrons (shown as :), from other ions or molecules.
Both the shared electrons come from the same atom.
The ion or molecule that forms the bond with the transition metal is called a ligand, e.g. :Cl -, :CN -, H2O:, :NH3.
More than one ligand can bind to a transition metal ion to form a complex ion.
The number of ligands so bonded is called the coordination number, for example:
coordination number 4: [CoCl4]2-,
coordination number 6: [CO(NH3)6] 3 +
Experiments
12.8.9 Chromium ions in solution, (See: 6.)

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1.1 Copper (II) sulfate solution with ammonia solution, ligand substitution
The copper ion, Cu 2 + (aq), in aqueous solution, forms a blue complex ion, [Cu(OH2)6] 2 +, with 6 water molecules.
The blue complex is the cause of the blue colour of copper sulfate solution.
Ammonia solution contains hydroxide ions, OH -, and is alkaline.
Add ammonia solution drop by drop to the pale blue copper sulfate solution.
A pale blue precipitate of insoluble copper hydroxide forms.
Cu 2 + (aq) + 2OH - (aq) --> Cu(OH)2 (s)
Add more ammonia solution to dissolve the copper hydroxide to form the deep blue complex ion, [Cu(NH3)4(OH2)2] 2 +.
Four of the 6 water molecules in the blue complex ion, [Cu(OH2)6] 2 + have been replaced by ammonia ions, leaving 2 water molecules still there.
So the number of ligands around the copper ion is still 6.

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1.2 Copper (II) sulfate solution with concentrated hydrochloric acid, ligand substitution
The copper ion, Cu 2 + (aq), in aqueous solution, forms a blue complex ion, [Cu(OH2)6] 2 +, with 6 water molecules.
The blue complex is the cause of the blue colour of copper sulfate solution.
Concentrated hydrochloric acid has a high concentration of chloride ions, which are better ligands than water,
because they are negatively charged ions and are attracted electronically to the copper ion, Cu 2 +.
Add concentrated hydrochloric acid drop by drop to the pale blue copper sulfate solution.
The solution turn yellow-green as the chloride ion ligands, Cl -, replace water in the complex ion.
Four chloride ions replace the 6 water molecules in the blue complex ion to form the yellow-green [CuCl4] 2- complex ion.
So the number of ligands around the copper ion has dropped from 6 to 4.

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1.3 Reactions of metals with ligands
See diagram 16.4.4: EDTA molecule
Metals and ligands form coordination bonds (coordination complexes), with both electrons coming from the ligand.
Ligands have a lone pair of electrons.
Metals do not have enough electrons to form covalent bonds by sharing one electron from the metal ion with one electron from the bonded atom.
The metals involved include Ag +, Al 3 +, Cu 2 + and Fe 3 +.
Examples of ligands include: -NH3, -OH2, -Cl -, -OCOCH3 -, -EDTA -4, -NTA -3.
Complexes include metal carbonyls, metal (CO)4, [Cu(H2O)6] 2 +, [PtCl4] 2-.
Metals usually bond with 4 to 6 ligands.
Chelates are ligands that bind more than one compound.
Copper forms a series of ligands with ammonia:
Cu 2 + + NH3 <--> CuNH3 2 +
CuNH3 2 + + NH3 <--> Cu(NH3)2 2 +
Cu(NH3)2 2 + + NH3 <--> Cu(NH3)3 2 +
Cu(NH3)3 2 + + NH3 <--> Cu(NH3)4 2 +
Ammonia is a monodentate (one tooth) ligand, because it forms one coordination bond with a metal.
Ethanediamine, (H2NCH2CH2NH2), is a bidentate (two tooth) ligand, because it forms two coordination bonds with a metal.
Triethanetetramine (trien) and nitrilotriacetic acid (NTA), are tetradentate ligands, because they form one four coordination bonds with a metal.
Ethanediaminetetraacetate (EDTA 4- ), is a hexadentate ligand, because it forms six coordination bonds with a metal.

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2.0 EDTA
EDTA, C10H16N2O8, Ethylenediaminetetraacetic acid, edetic acid
EDTA, C10H14N2Na2O8.2H2O, Ethylenediaminetetraacetic acid disodium salt, Toxic if ingested
EDTA, ethylene diamine tetra acetic acid (HOOC.CH2)2N(CH2)2N(CH2.COOH)2
See diagram 16.4.4: EDTA molecule
1. Ethylenediaminetetraacetic acid (HOOCCH2)2N(CH2)2N(CH2COOH)2, edathamil, sequestering agent for metal ions, Mg 2 +, Ca 2 +.
Complexing agent for most metal ions, especially Ca 2 + and Mg 2 +.
Used for water softening, remove scale from kettles, medical removal of excess metallic ions.
If a ligand is defined as a small molecule that binds to a larger molecule, then chelates can be said to bring about the complexation of a ligand.
The terms ligand, chelate, chelating agent and sequestering agent are used in slightly different ways in chemistry, medicine, and general industry.
If EDTA = H4Y, then the disodium dihydrate form = Na2H2Y.2H2O
H2Y 2- + Ca 2 + <--> CaY 2- + 2H +
2. EDTA disodium salt, ethylenediaminetetraacetic acid disodium salt dihydrate, C10H14N2Na2O8.2H2O, EDTA disodium salt,
ethylenediaminetetraacetate dihydrate (HOOCCH2)2N(CH2)2N(CH2COONa)2.2H2O, (0.1 M, solution + sodium azide), for electrophoresis, molecular biology.
3. A chelate is a metal ion bound to two or more atoms of a chelating agent (sequestering agent),
For example, the simple chelating agent 1,2-diaminoethane (ethylene diamine), NH2.CH2.CH2.NH2, forms bonds to a metal ion through its nitrogen atoms.
[Diaminoethane, ethylene diamine, C2H4(NH2)2, ligand, chelating agent, Toxic by all routes, Corrosive]
Porphyrin chelates include haeme, in haemoglobin, bonded to iron (II) ion, and chlorophyll bonded to Mg (II) ion.
Similarly vitamin B-12 has cobalt (II) ion bonded to a chelating agent.
4. The synthetic chelating agent EDTA can form complexes with calcium and magnesium ions.
So it can form the calcium complex [Ca(EDTA)] 2-.
The sodium salt used as an antidote for metal poisoning, an anticoagulant, enzyme deactivation, bactericide, industrial processes.
EDTA disodium salt,   (HOOC.CH2)2N(CH2)2N(CH2.COO.Na)2.2H2O.
EDTA deactivates the enzymes containing metal ions that cause food spoilage, loss of colour and loss of flavour.
EDTA dissolves the calcium carbonate scale caused by hard water and prevents stored blood from clotting by sequestering calcium ions.
Calcium disodium EDTA is a treatment for lead poisoning by exchanging its chelated calcium for lead so lead chelate is excreted.
5. Industrial synthesis of EDTA
NH2.CH2.CH2.NH2 + 4 H.CHO + 4 Na.CN + 4 H2O
(Na.OOC.CH2)2N(CH2)2N(CH2.COO.Na)2 + 4 NH3
1, 2-diaminoethane (ethylenediamine) + methanal (formaldehyde) + sodium cyanide + water -->
sodium salt + ammonia (Na.OOC.CH2)2N(CH2)
2N(CH2.COO.Na)2 + 4 HCl -->
(HOOC.CH2)2N(CH2)2N(CH2.COOH)2 + 4 NaCl
sodium salt + hydrochloric acid --> EDTA + sodium chloride
6. Chelating agent
A chelating agent binds to metals and prevents them from participating in chemical reactions.
Calcium disodium EDTA, C10H12CaN2Na2O8, is an odourless crystalline powder, light salt flavour, food additive, preservative, flavoring agent.
It is used in sauces, canned food products and cosmetic prodyucts to preserves texture, flavor and colour, increase shelf life, enables foaming
It prevents metals accumulating, prevents discoloration and is used to treat metal toxicity, e.g. lead or mercury poisoning.
Eriochrome black T (metal indicator for the titration of calcium and magnesium ions with EDTA)
1,2-diaminoethane, C2H8N2, C2H4(NH2)2, ethylenediamine, smells like ammonia, related to EDTA, Toxic by all routes, corrosive
1,2-diaminoethane, Solution < 1%, Not hazardous
EDTA, Ethylenediaminetetraacetic acid, edetic acid, [(HOOCCH2)2NCH2], C10H16N2O8,
A chelating agent that binds calcium and heavy metal ions including lead, so used to treat lead poisoning, food additive, Anticoagulant, EDTA disodium, C10H14N2Na2O8.2H2O, edetic acid disodium salt.
3.3.7 Prepare nylon polymer
19.1.22 Sequestrants, food additives

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2.1 Mineral deficiency experiment, hydroponics, EDTA
To make four kinds of culture solutions based on large elements, trace elements and iron solution.
Among them, the mother solutions of the iron solution and trace elements may be kept in the refrigerator.
The main elements are compounded in two kinds of reserve solutions (A and B).
The table of the prescription of reserve solution, unit: gram / litre water
Table 9.9.18.5

Solution	Chemical Name	Molecular Formula	Volume g / litre
A, main	calcium nitrate crystals	Ca(NO3)2.4H2O	30
"	potassium nitrate (nitrate of potash)	KNO3	.
B, main	ammonium phosphate (di-ammonium hydrogen phosphate)	(NH4)2 HPO4	25
.	magnesium sulfate (Epsom salts)	MgSO4.7H2O	25
C, iron	EDTA, disodium salt (disodium ethylene diamine)	EDTA-Na2	2.682
.	ferrous sulfate (iron (II) sulfate-7-water)	FeSO4.7H2O	.
D, trace	boric acid (boracic acid)	H3BO3	2.86
.	zinc chloride	ZnCl2	0.22
.	copper (II) sulfate crystals	CuSO4.5H2O	0.08
.	molybdic acid	H2MoO4	0.02
.	manganese chloride	MnCl2	0.08

Dissolve EDTA disodium salt in hot water, then add iron (II) sulfate-7-water and mix while it is hot.
Do not mix the Solutions A and B to avoid precipitating calcium sulfate.
Keep Solutions A and B in a shaded place.
Keep Solutions C and D in the refrigerator.
In Solution A the proportion of calcium nitrate and potassium nitrate can be adjusted depending on the water quantity and the variety of plants at the place in the locality.
For example if hardness of water is great reduce the quantity of calcium nitrate.
Make up the solutions with deionized water and regulate pH to 5.5 to 5.8 with HNO3 or KOH according to the needs of different vegetables, e.g. if the leaf should has the high content of nitrogen increase the content of phosphorus and potassium after the flowering phase.

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2.2 Tests for metal ions in water, EDTA chelates
The different forms of hardness are expressed as "calcium carbonate hardness".
EDTA is [ethylene diamine tetra-acetic acid, HOOCCH2)2N(CH2)2N(CH2COOH)2].
The disodium salt of EDTA combines with metals to form chelates.
Chelates are compounds where a multidentate ligand, e.g. as an enolate anion of a diketone, is bound to a central atom of a coordination complex.
EDTA is a complexone and is used in special soaps to remove metal contamination and as a chelating agent for analytical determination of metal contamination.
It is available as "EDTA (0.5 M)", which is a concentrated volumetric solution for dilution to prepare 1 litre of 0.5 M standard solution.
Experiment
1. Prepare a 0.01 M EDTA solution by dissolving 1.861 g in 500 mL water.
2. Prepare pH 11 buffer solutions by adding 7.0 g ammonium chloride solution to 57 mL of concentrated aqueous ammonia solution.
Dilute to 100 mL.
BE CAREFUL!
3. Prepare an approximate 0.01 M solution MgCl2.6H2O by dissolving 2 g in 1 litre.
4. Eriochrome Black T or Erio T indicator is the indicator for determining Ca 2 +, Mg 2 + and Zn 2 + with EDTA.
Dissolve 0.2 g EDTA powder in aqueous ammonia solution.
Pour 100 mL of the water to be tested in a conical flask on white paper.
Add 1.0 mL of the Mg 2 + solution, 3 drops of "Erio T" indicator solution and 1 mL of the buffer solution.
Add drops of EDTA solution until a blue colour appears.
1 mL EDTA solution / 1.0 mg CaCO3.
Ca 2 + + H2EDTA2 <--> CaEDTA 2- + 2H +.

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2.3 Tests for water hardness, EDTA titration, Calmagite indicator
The usual form of EDTA used for measuring water hardness is Na2EDTA.2H2O, the ethylenediaminetetraacetic acid disodium dihydrate salt.
In this ion exchange reaction, the EDTA is in the sodium form and only the cation Ca 2 + is removed.
Let RZ = the ion exchange resin.
2RZ-SO3 - Na ++ Ca 2 + <--> (RZ-SO3 - )2Ca 2 + + 2Na +
Make the following solution: 5.0 mL of water sample, 1.0 mL of 1.5 M NH3 / 0.3 M NH4Cl buffer, 0.1 mL of 2% ascorbic acid solution, 1.0 mL of 0.01 M Na2MgEDTA / 0.1 M NH3 solution, 3 drops of
0.1% Calmagite indicator.
The buffer keeps the hydrogen ion concentration of the solution at the optimum value.
The ascorbic acid solution prevents oxidation of the indicator.
Titrate the solution with 0.01 M Na2EDTA (Na2EDTA.2H2O) until the colour of the solution changes from red to blue.
Repeat with a blank titration 5.0 mL of deionized water instead of the water sample.
Subtract the volume the blank titration from the volume of the water sample titration.
1. Calculate the water hardness in units of millimoles per litre, i.e. the sum of the calcium and magnesium ion concentrations.
2. Calculate the water hardness in units of parts per million of CaCO3 (ppm, 1 ppm = 1 mg per litre), i.e. milligrams of CaCO3 per litre, assuming that the ions titrated came from CaCO3.
Magnesium usually also occurs in hard water, but this second calculation is often used by water engineers for convenience.
(mg CaCO3 / L) = (mmol Ca 2 + + Mg 2 + / L) × (1 mol CaCO3 / mol Ca 2 + + Mg 2 + ) × (100 g CaCO3 / mol CaCO3).

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2.4 Tests for water hardness, EDTA titration
Eriochrome, C20H12O7N3Na, solochrome black, toxic if ingested
1. Use Eriochrome Black T indicator and titrate with EDTA.
Check that the pH is 10 when adding the buffer, using a pH meter.
Use Eriochrome black T indicator that has been ground with salt rather than a liquid.
EBT is blue in a buffered solution at pH 10.
It turns red when Ca 2 + ions are added.
At the blue end-point, sufficient EDTA has been added, the metal ions are chelated by EDTA, to leave the free indicator molecule.
Eriochrome Black T, C20H12N3O7SNa, is blue in protonated form, but turns red when forming a complex with Ca 2 +, Mg 2 +, and some other metal ions.
EDTA, Ethylenediaminetetraacetic acid, aminopolycarboxylic acid, [CH2N(CH2CO2H)2]2.
2. Hardness Ratings (mg CaCO3/L equivalent): < 15 very soft; 15-50 soft; 50-100 medium hard; 100-300 hard; > 300 very hard
3. The quantity of hardness ions will be determined by titration.
EDTA, a weak acid, will be used as the titrant.
In its ionized form, it is able to form soluble complexes with calcium and magnesium cations.
The indicator added to the sample is Eriochrome Black T.
Initially, the indicator will form a complex with the cations.
When complexed it is red in colour.
As the EDTA is added dropwise to the sample, it replaces the Erio T and forms more stable complexes with calcium and magnesium.
When the indicator is released by the metal ions, it has a distinct blue colour.
Therefore, the endpoint of the titration is marked by the colour change from red to blue.
To determine the hardness of water by measuring the concentrations of calcium and magnesium in water samples by titration.
The titration should be completed within 5 minutes of buffer addition.
Some metal ions may interfere in the titration by causing fading or indistinct end points.
Experiment
1. Clean a 25 mL volumetric pipette by rinsing it with 10% HCl, then 2 washes of distilled water.
Hold the pipette horizontally and rotate it so that the liquid washes all the inside surfaces.
Place the pipette on a towel.
Label all glassware.
Clean and fill the burette
2. Titrant solution: Pour about 100 mL of 0.01M EDTA titrant solution into a 250 mL beaker
3. Analysis solution: Pipette 25 mL of the water sample into a conical flask and dilute it with 25 mL distilled water.
Add at least one mL of pH 10 buffer solution to the sample.
The pH should be 10, so check the pH with pH paper or a pH meter.
4. Add a 1-2 drops Eriochrome Black T indicator (or a small scoop of powder indicator formulation) to the conical flask.
The solution should now be red / pink.
5. Titration: Test 1. Immediately begin to titrate the sample two drops at a time.
Be careful to titrate slowly near the endpoint, as the colour will take about 5 seconds to develop.
Thus, add the last few drops at 3-5 second intervals.
The endpoint colour is blue.
6. Perform this procedure at least two more times, Test 2 and Test 3.
The titre volumes should be within 0.1 mL of each other.

Titre (mL)	Test 1	Test 2	Test 3
Initial Volume	-	-	-
Final Volume	-	-	-
Titre Volume	-	-	-

Express hardness as parts per million (mg per litre) of equivalent CaCO3.
If the titration required 5 mL EDTA, the calculation is as follows:
(5 mL 0.01 M EDTA / 0.025 L sample) x ( 1 mg equivalent CaCO3 / 1 mL 0.01 M EDTA) = 200 ppm CaCO3
Reference: Standard Methods for the Examination of Water and Wastewater, 20th ed. L. S. Clesceri, A. E. Greenberg, A. D. Eaton
editors, 1998, American Public Health Association.

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2.5 Ion exchange resins, deionized water
Let RZ = the resin, an organic polymer matrix.
Charged groups are bound to the resin.
Cation exchange resin, H + form, to remove cations, e.g. Ca 2 +, from solution.
2RZ-SO3 - H ++ Ca 2 + <--> RZSO3 - )2Ca 2 + + 2H +
Anion exchange resin, OH- from, to remove anions, e.g. Cl -, from solution
RZ-N(CH3)3 + OH - + Cl - -->RZ-N(CH3)3 + Cl - + OH -
To "soften" water, usually only a cation exchange resin is used.
If both a cation exchange resin and an anion exchange resin are used with tap water to remove ionic salts by ion exchange, the resulting solution is deionized water, whic is a cheaper alternative to distilled water.

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Combination and decomposition
* Combination or synthesis means to bring two or more elements together to form a compound; decomposition means to break down a compound into two or more elements (ie. they are the opposite of each other)
* Useful combination reactions:
1. Hydrogen-oxygen fuel cells: a spontaneous combination reaction between H2 and O2 to form H2O and in doing so, energy is released (which can be harnessed as an electric current)
2. Haber process: combining N2 and H2 to form NH3 (Ammonia) à essential for making fertilizers (the basis of agriculture) and explosives
3. Combination of Mg and O2 to form MgO that in doing so, releases a white light à the original ‘flash’ for photography
* Useful decomposition reactions:
Hoffman’s apparatus uses an electric current to reverse the process and re-split the water into each element to then be re-used.
Most hydrogen is produced from fossil fuels today, so one application of the decomposition of water is as a route to producing clean hydrogen.
Hydrogen is a fuel that produces no carbon emissions when it burns - for hydrogen-powered vehicles.
Making hydrogen in this way requires electricity for electrolysis.
The problem is that the hydrogen water electrolysis is only environmentally benign if the electricity you’ve put into it is not made from burning fossil fuels.
We cannot plug a beaker of water into a standard electricity socket and claim we’ve made ‘green’ hydrogen if that socket is powered by coal-fired power stations.
It is possible, however, to do the same decomposition reaction and make hydrogen while plugged into a solar panel or a wind turbine.
On the Orkney Islands, they power several zero emission vans in this way through electrolysis driven by excess wind energy from its turbines.
Decomposition of hydrogen peroxide H2O2 --> O2 + H2O
This process releases free radicals that destroy molecules or bacteria (eg. bleach, cleaning).
(Blood speeds up this process as a catalyst!)
Demonstrations
* Hoffman’s apparatus – decompose H2O into H2 and O2 then reform H2O by lighting a splint/ match with H2 that is collected (reacts with O2 in the air) à basis of fuel cells
* Combination of 2Mg + O2 à 2MgO (light Mg in Bunsen burner: DO NOT LOOK DIRECTLY AT THE FLAME)
* Decomposition of hydrogen peroxide (with MnO catalyst/ liver enzyme)
Displacement reactions
The word displace means to push out.
A more reactive element can displace a less reactive element out of its compound (e.g. solution) during a chemical reaction.
Displacement reactions can be used to investigate the reactivity of metals and extract metals from metal oxides.
Useful displacement reactions
Thermite Welding: A mixture of Al and Fe2O3 reacts to produce molten iron that is used to join railway rails together.
2Al + Fe2O3 --> Al2O3 + 2Fe
Al replaces Fe in Fe2O3 to form Al2O3
Steel Making: We can extract iron from its ore (Fe2O3) by heating it with carbon in giant blast furnaces.
3C + 2Fe2O3 -->4Fe + 3CO2
C replaces Fe in Fe2O3 to form CO2.
Extraction of Metals: Heating a mixture of Cr2O3 and finely divided C produces metallic chromium.
3C + 2Cr2O3 -->4Cr + 3CO2
C replaces Cr in Cr2O3 to form CO2 and chromium metal.
Acid Indigestion: We can relieve the distress when the stomach produces too much HCl by drinking a solution of sodium hydrogen carbonate.
HCl + NaHCO3 -->NaCl + H2CO3
The H in the HCl displaces the Na in NaHCO3 to produce H2CO3
Experiments on metal displacement
1. Place small samples of copper, zinc, magnesium, silver in each of their respective solutions (replace Lead with Silver)
Observe if the metal ion is displaced from the solution by a more reactive metal.
2. Extract silver from solution by displacement by solid copper wire (or tree)

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19.4.2 Electrophoresis, food dyes, marking pen ink
See diagram 19.2.1.1a: Electrophoresis.
Electrophoresis is the movement of molecules in a solution under constant electric charge.
Electrophoresis is the movement of colloidal particles in a fluid caused by an electric field.
If a solution contains different sized molecules with different charges, the molecules can be separated, because they move at differnt speeds under constant electric charge.
Electrophoresis can used to separate different proteins in solution.
Gel electrophoresis is used to sort molecules based on their size and charge.
An electric field is applied to make molecules move through an agar gel to make negatively charged molecules move towards the positive terminal and positively charged molecules move towards the negative terminal.
Larger molecules move slower than smaller molecules leaving the different sized molecules as bands the gel.
Experiments
1. Cut the sides of a 10 cm × 5 cm flat bottom plastic container down to 3 cm height, e.g. a margarine container.
Fold a piece of aluminium foil over one short end of the container to cover both the outside end and extend to the bottom of the container.
Do the same at the other end of the container.
2. Make a comb from a piece of flat thick plastic, e.g. the lid of an ice cream container for a thin comb or a Styrofoam meat tray for a thick comb.
The comb must fits neatly into the width of the plastic container.
It has two lips that hang over the sides of the plastic container tub to keep the comb in place.
However, the teeth of the comb should not touch the bottom of the plastic container.
Cut 6 teeth in the comb.
Each tooth should be 5 mm wide and 15 mm long.
3. Prepare a 0.1% bicarbonate buffer by dissolving 0.2 g of sodium bicarbonate in 200 mL of water.
Mix 1g of agar in 100 mL of the 0.1% bicarbonate buffer and heat to boiling in a microwave oven.
Heat for 30 seconds then 10 second pulses until it boils.
Leave to cool to hand temperature.
4. Make 1 cm diameter spots of vegetable food dyes, e.g. cochineal or ink from coloured marker pens on filter paper.
5. Prepare a 1% agar gel solution by dissolving 1 g of agar in 100 mL in bicarbonate buffer solution.
Fill the plastic container with agar gel to a depth of 1 cm.
Insert the comb so that the top of the agar solution is just below the top of the teeth of the comb.
Fix the comb 2 cm from one end of the plastic container.
Leave the gel to set undisturbed for 15 minutes.
When the gel is set, carefully remove the comb.
6. Cut out 3 mm × 4 mm rectangular pieces of the colour spots and insert them into the wells formed from the teeth of the comb.
Pour 100 mL of bicarbonate buffer solution into the plastic contained to completely cover the gel.
Some colour from the paper rectangles may diffuse into the buffer solution, but this will not affect the colours diffusing through the gel.
7. Connect the gel to five 9-volt batteries connected in series with wire leads and alligator clips.
Connect the end of the tank with the samples to the negative terminal of the battery.
If fewer batteries are used the samples will take longer to run and may diffuse into the gel.
Leave the circuit connected for 45 minutes until separation of samples occurs.

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