The 3 Rs - Keratin

Objective: observing textile fibres under a microscope

Materials: microscope slide, coverslip, tweezers, scalpel, textile fibres (cotton, wool, silk)

Solutions: distilled water, Lugol’s solution

Procedure

1. Unthread a tissue sample with a scalpel and tweezers so that you get a few isolated fibres.
2. Place these on a microscope slide, add a drop of water or reagent and cover with the coverslip.
3. Place the slide on the stage of the microscope, focus and observe.

Results

The students can describe what they see and identify the origin of the fibres using the diagram.

You can download the identification sheet here as docx and as pdf file.

Materials

• Rough wool fibres. The wool must be untreated, raw and undyed to avoid the treatments altering the results.
• Sodium hydroxide (NaOH), use a 1.0 mol/L (M) solution. If time permits, wait for 2 hours before the extraction of the keratin. If it is not possible to wait, a period of 30 minutes already allows you to have a sufficient result.
• Beaker
• Glass rod
• Gloves
• Lab coat
• Protective goggles

Method

Safety first: Wear protective gloves, a lab coat, and goggles.

Put the wool fibres in a beaker. Cover them with the sodium hydroxide solution and mix the mixture with the glass rod. Make sure that the wool is completely covered by the extraction solution. Let it stand for a few hours.

The indicated time (ideally 2 hours or at least 30 min) refers to the hypothesis of a 1.0 M solution; alternatively, a less concentrated solution (0.5 M) can be used and of allowed to rest overnight.

Extract a part of the fibres from the extraction solution and observe their appearance.

You can plan various lab experiments with the students using different solutions and different times for the extraction. The students can compare the results.

Results

• Appearance of the wool before extraction: White colour, soft, crimps or not

• Appearance of the wool after the extraction period: The wool takes on a dark colour and a gelatinous appearance.

• Appearance of the extraction solution at the end experience: The clear and colourless solution at the beginning becomes dark and cloudy.

Discussions

1. Do you think that a chemical reaction has taken place that has somehow modified the wool?

The changes observed in appearance of the fibres, both in terms of colour change and turbidity of the solution, indicate that chemical reactions have occurred.

2. Can you make assumptions as to what kind of changes could have occurred at the molecular level?

By extracting the keratin with NaOH, denaturation occurs with a partial degradation of the protein.

Keratin can be extracted without degrading it, but the extraction process is rather long, and therefore a difficult proposition in laboratory setting. Non-degradative extraction is carried out using sodium metabisuphfite, which breaks the sulphur bridge bonds, and urea (denaturing agent).

After extracting the keratin with a solution of NaOH, the present protein will be flocculated in the solution, using different substances also in common use.

By decanting and drying you can obtain the keratin powder. Another method is centrifugation, which is faster but a centrifuge is needed. Decanting and drying is slower but can be done in every school.

Materials

• Keratin solution in NaOH
• Ethanol (CH₃CH₂OH)
• Acetone (Dimethylketone, CH₃-CO-CH₃)
• Citric acid 6% (Ac. 3-carboxy-3-hydroxy-1,5-pentanedium, C₆H₈O₇)
• White wine vinegar
• Lemon juice
• Pipettes
• Test tubes
• Petri dish
• Protective goggles
• Protective gloves

Method

Safety first: Wear protective gloves and goggles.

Strain the lemon juice. It is necessary to filter the lemon juice to try to reduce the turbidity.

Citric acid in 6% solution has a pH value similar to that of lemon, therefore, you can use a citric acid solution instead of lemon juice.

We have chosen to use both to show that substances that appear daily on our dinner table (lemon, vinegar), can be used as chemical reagents. It is also interesting to observe the layering of the protein with lemon juice.

Take five tubes, mark them and add about 10 mL of different solutions in each one: ethanol, acetone, citric acid, vinegar, lemon juice.

Add about 1-2 mL of the extraction solution drop by drop to each tube. Enter the observations in a table.

Then pour the contents of each tube into a petri dish and let it evaporate until it is completely dry. Observe against a dark background and report your results in a table.

Appearance of the solutions

Appearance after complete drying:

Discussion

1. What does the term flocculation mean?

Chemical-physical process in which the solid phase of a colloidal system tends to separate forming flakes in suspension.

2. What is the explanation for the behaviour of keratin in different solutions?

Precipitation (or protein flocculation) can be carried out in different ways:

1. Induced by pH: by changing the pH of the solution, for example by adding acetic acid, to get close to the isoelectric point (pI or pHi) of the protein.
2. Solvent variation: the dielectric constant of a solvent has an influence on the electrostatic interactions between protein, leading to flocculation (hydrogen bond formation). A weak dielectric constant (such as in ethanol or acetone) tends to reduce the solubility of a protein.

The behaviour is different if flocculation is induced by pH or by solvent variations.

Preparation of the keratin

Safety first: Put on protective goggles, a lab coat and gloves.

1. Weigh about 5 g of raw wool into a beaker and introduce 150 mL of 1 mol/L NaOH.

2. Extract the keratin as described in the part Extraction of keratin.

3. Transfer the extract into an Erlenmeyer flask by filtration.

4. Under a hood, pour acetic acid (CH₃COOH) drop by drop into the extract until precipitate crystals are observed (keratin flocculation).

5. Separate the keratin crystals from the solution in which they are contained by centrifugation. If you do not have a centrifuge at school, you can separate the solid part from the solution by decantation as described in the part Flocculation and precipitation of wool keratin. The decantation process is slower than centrifugation.

6. Transfer the keratin to a watch glass.

7. Allow to dry.

Keratin conditioner

For 50 mL of the product, weigh into a beaker:

• 7.5 g of a 25% m/V keratin solution in glycerine,
• 3 g Cetyl alcohol,
• 7.5 g Shea butter,
• 7 g Almond oil and
• 7 g Citric acid (preservative).

Place the beaker in a bain-marie and heat until the ingredients have melted.

Add 1-2 drops of essential oil.

Transfer the resulting product into a container.

• Plant: Curcuma longa
• Part used for the tincture: crushed root
• Colouring: curcumin

Materials

• Water
• Beaker or pot
• Wool or other natural fibres

To prepare the dye, the chopped turmeric root must be used in an amount equal to 50% of the weight of the dry yarn.

Procedure

• Take the wool, weigh it and immerse it in water at room temperature (use the beaker or a pot of water). Use water that can cover the wool.
• To prepare the dye, the chopped turmeric root must be used in an amount equal to 50% of the weight of the dry yarn. Otherwise, turmeric powder can be used directly in an amount equal to 5-10%.
• If you have only turmeric powder available, you have to calculate the weight of the powder in relation to the wool to be dyed.
• Dissolve the powder in water.
• Add the wool.
• Boil everything for 20-30 minutes.

• Plant Brassica oleracea convar. capitata var. rubra
• Part used for dyeing: leaves
• Dye: anthocyanin

The weight of the cabbage must be about 60 times the weight of the tissue.

Materials

• Gloves
• Red cabbage
• Alum powder (hydrated double aluminium potassium sulphate salt)
• Potassium hydrogen tartrate (Rock alum (aluminium potassium sulphate, which is a real etchant) is used at 10-15% by weight and potassium hydrogen tartrate which softens the hardened wool from the etching by 5% The percentages refer to the dry wool to be treated. That is, 100 g of dry wool requires 10-15 g of alum and 5 g of potassium hydrogen tartrate.)
• Water
• Washed wool
• Stainless steel pot
• Stainless steel mixing bowls

Mordanting the wool

This process acts on the wool, or generally on the natural fibre. The water-soluble dye substances in the bath are fixed permanently and indelibly on the fibres.

The process is known as mordant dyeing. It is the most important process of preparing fibres to accept colour – it helps to ensure the most durable and long-lasting colours. In the case of red cabbage, if you don’t mordant the material you are planning to dye naturally, the colour will wash out when you finish the dye process.

This is done as follows:

1. Soak the previously washed wool in water three minutes before starting the
2. Weigh alum and cream of tartar in the above proportions.
3. Put about 200 mL water to heat in a small
4. Add the 15 g of alum and 5 of cream tartare in the 200 cc of water and stir to dissolve.
5. Fill 2-3 l of water for every 100 g of dry wool into a larger pot, add the etching solution and immerse the wet wool.
6. Simmer for 1 hour and stir frequently.
7. Allow the wool to cool, slowly and naturally, by leaving it in the etching solution.
8. Rinse the wool very well with cold or warm water.

Remember that the containers used must be made of stainless steel. Aluminium is not recommended as it can alter colours.

Procedure for dyeing

Cut the cabbage into thin strips. Heat a pot of water to 60°C.

Place the cabbage in the pot and warm up for about 30-40 minutes to extract anthocyanins from the vegetable. If the water begins to boil, take the pot off the heat for a moment. Boiling the cabbage will destroy some of the potential dye!

• Strain the cabbage cooking liquid and pour it into another pot.
• Place your mordanted wool in the pot containing dyed water, gently pushing down on the wool to submerge it. Carefully squeeze the wool in the pot to make sure all the fibres absorb the dye water.
• Set the heat to medium-low, letting your wool soak in the pot for an hour - maybe longer if you want to try getting a deeper colour. It’s very important you don’t boil the water during this step.
• Then remove the wool from the colour bath and rinse it gently.
• Spread and let the wool dry overnight.

The colour obtained from cabbage varies according to pH and tissue.

At acid pH, a reddish colour occurs, while at basic pH, a greenish blue colour occurs.

In our case, the wool becomes light purple as you can see in the photo.

In recent years, the various applications of wool have been studied. In particular, it has been seen that wool might be used to clean up soils contaminated by heavy metals. Its use in this sense would have a dual purpose: to help in the disposal of excess or waste wool, and to use a completely natural material.

Material needed

• Wool
• Copper sulphate solution (CuSO 4(aq) 200 mg/l)
• 2 Copper test strips as required
• 2 Beakers
• Glass rod
• Gloves
• Protective lab coat
• 7 test tubes
• Copper assay strips

Procedure

Safety first: Wear protective goggles and a lab coat

Pour 20 mL of the copper sulphate solution into 2 beakers.

One beaker will be the reference. Add a piece of wool into the second beaker and stir so that it is soaked by the solution and wait a few hours.

Observe any colour variations in the wool and in the solution.

The white colour of the wool allows the adsorption of Cu²⁺ ions to be noted.

Use a copper assay strip to quantify the amount of copper in each beaker.

Put the strips into the solutions, wait about 20 seconds and observe the colouring.

Thanks to the change in colour of the different solutions, the change in copper concentration in the presence of the wool can be determined.

Results

In the beaker without the wool you can obtain an intense violet colour on the strip, indicating a high copper concentration.

In the beaker with the wool you can obtain a red colour on the strip, indicating a low copper concentration.

• Solutions: Copper sulphate solution
• Strip colouring: Violet
• Solution concentration: 200 mg/l

• Solutions: Solution of copper sulphate and wool
• Strip colouring: Very light pink
• Solution concentration: 10-20 mg/l (the colour and consequently the concentration also depend on the reaction time)

To measure the amount of copper in the solution, you can proceed using semi-quantitative test strips for copper ions, and prepare a colorimetric series as follows.

Using the copper(II) sulphate solution provided, prepare five tubes of diluted copper(II) sulphate, according to the following table. Ensure the solutions are well mixed.

Pour a 10 cm³ sample of the copper solution from your two beakers (Experiment A, the beaker with the wool and the other one without the wool) into two other test tubes.

Compare the colour of your tube from the experiment A (absorbing Cu²⁺ from wool) with those from the colorimetric series. Which one matches the colour best?

Discussion

1. What colouring did the wool take on after being soaked in copper sulphate? Why?

The wool appears grey/blue in colour because it has adsorbed Cu²⁺ ions. 

2. Calculate the amount of copper absorbed by the wool during the incubation time.

The calculation can be made approximately by comparing the colours of the strips.

3. Do you think that time is a variable to be taken into account when assessing the wool’s absorbing capacity? How could you verify this?

Repeat the experiment, dipping different strips into the solution at different times.

4. Design an experiment to measure the amount of wool needed to absorb a specific amount of copper, or repeat the experiment, using beakers with different amounts of wool, previously weighed in the same concentrations of copper.

1. Extraction of keratin

2. Hair conditioner

3. Dyeing wool with tumeric

4. Dyeing wool with red cabbage