Applied Science BTEC Nationals/Chemical Laboratory Techniques/Kastle-Meyer
- By the way, 'Phenolphthalein' is pronounced 'fee-nolf-THAY-leen'.
A dry sample is collected with a swab or filter paper. First a few drops of ethanol, then a few drops of reduced phenolphthalein reagent solution, and, finally, a few drops of w:hydrogen peroxide are applied to the swab. If the swab turns pink then it is a positive test. This test is nondestructive to the sample, which can be kept and used in further tests at the lab. This test has the same reaction with human blood and animal blood so further testing would be required to determine which one it is.
Preparation of reduced phenolphthalein 
The phenolphthalein used in this test has been modified from its conventional form, in that it has been reduced by two electrons and is pre-dissolved in alkaline solution. This is typically achieved by boiling an alkaline solution of phenolphthalein with zinc powder, which acts as the reducing agent. Upon reduction, the very intense pink colour of the cationic form of phenolphthalein fades to a faint yellow colour. It is this form of phenolphthalein that is present in Kastle-Meyer test kits. In order to generate the intense pink colour indicative of a positive test, the reduced phenolphthalein must be oxidised back to its normal, coloured form.
Mix the following reagents in a 250 cm3 round-bottomed flask:
- Phenolphthalein 2.0 g
- Potassium Hydroxide 20.0g
- Deionised Water 100 cm3
- Zinc Dust 20.0 g
- A few anti-bumping granules
Boil under reflux for 2-3 hours until the solution has lost its pink colour. Cool and decant into a bottle containing some zinc to keep in the reduced form. Stored in a fridge, in the dark, the shelf life is 6 months.
Alternative mixtures 
The preparation can be scaled-up and the low-concentration solution with ethanol created in one step:
- Phenolphthalein 4 g
- Sodium Hydroxide pellets 40 g
- Zinc dust 20 g
- Deionised water 1000 cm3
- Ethanol to bring the total volume up to 1200 cm3
Reflux in a 5000 cm3 round-bottomed flask. After reflux, restore to 1200 cm3 with ethanol.
Another method uses similar proportions but avoids refluxing:
In a 1 dm3 beaker, add
- Phenolphthalein 1 g
- Sodium Hydroxide pellets 10 g
- Zinc dust 5 g
- Deionised water 250 cm3
Using a stirring hot plate, mix and heat until the solution loses its pink colour. Do not boil. This process may take 2 to 3 hours.
Decant the liquid into a 500 cm3 measuring cylinder. Add ethanol to make 300 cm3 of solution.
Add a small amount of zinc powder to a brown bottle, and pour the phenolphthalein solution into this bottle. Label, date, and store the bottle in a refrigerator.
Safe disposal of zinc 
Zinc powder or dust in contact with water or damp air evolves hydrogen. The heat of the reaction is sufficient that the hydrogen may ignite. Therefore the zinc should not be discarded in the wastebasket. The following disposal procedure should be followed for less than 20 g of zinc:
Follow standard laboratory procedures of wearing gloves and safety aprons. Add dilute hydrochloric acid to the zinc dust in a beaker. Allow bubbles to form and slowly add more acid till no more bubbles are seen.
When all the zinc has dissolved add sodium carbonate solution to the mixture. Bubbles will be formed. Keep on adding the sodium carbonate small quantity at a time till the zinc precipitates as zinc carbonate.
The zinc carbonate can be now filtered and disposed of as it is non-toxic.
Working Solutions 
Ethanol 10 cm3
Reduced phenolphthalein stock 2 cm3 Distilled Water 10 cm3 Ethanol 2 cm3
(Or use the alternative mixture noted above with no extra water or ethanol).
3% Hydrogen Peroxide 10 cm3
- Using a piece of filter paper or paper towel, rub the stain suspected to be blood to collect a sample. If the stain is dry, you may moisten the paper slightly with distilled water. You will not see a visible blood stain on your filter paper.
- Lay the paper out so that the spot that you rubbed is exposed.
- Two or three drops of Ethanol (solution 1) are placed on the stain.
- Two drops of working phenolphthalein solution (solution 2) are added to the stain.
- After waiting to ensure that no colour develops at this stage, two or three drops of 3% Hydrogen Peroxide (solution 3) are added.
- An intense pink colour is a positive test for peroxidase activity, indicative of haemoglobin. This is not a confirmatory test for blood.
- A positive reaction is indicated by the development of a pink colour within 5 seconds. Reactions occurring after 5 seconds, or before the addition of the hydrogen peroxide are inconclusive. A pink colour after phenolphthalein has been applied but before hydrogen peroxide has been applied normally indicates a false positive due to an oxidising agent being present. Rust could cause a false reading of this type.
Standards and Controls: Standards should include a known blood stain (positive control) and a known blood-free sample (negative control). These controls will be run before analysis and recorded in the laboratory notes.
In the relevant reaction, hydrogen peroxide reacts with the haemoglobin in the blood, with the phenolphthalein not participating in this first process. In its reaction with hydrogen peroxide, the haem centre of haemoglobin undergoes the following O-O bond w:homolysis reaction:
- HOOH + Fe3+[haem] → Fe4+=O[haem] + OH· + H+
The products of this reaction are one equivalent each of a high-valent iron-oxo species and hydroxyl radical, either of which can oxidise the reduced phenolphthalein back to its coloured form. The amount of acid produced in this reaction is insignificant in comparison to the concentration of base present in the phenolphthalein reagent solution. In addition, this reaction of haem with peroxide is catalytic, making this test very sensitive to small quantities of blood present on the test swab.
This test has some limitations. Namely, the enzymes in some vegetables (especially tomato, potato, cucumber, horseradish) can cause a false positive test result. Other non-blood substances which give positive results are: some fruit extracts, some metallic substances, or any other peroxidase-like substances.
Adapted from w:Kastle-Meyer test.