Thymolphthalein: Characteristics, Preparation And Applications

The thymolphthalein is a pH indicator with the scientific name: 3,3-bis [4-hydroxy-2-methyl-5- (1-methyl) phenyl] -1 (3H) -isobenzofuranone and its chemical formula is C 28 H 30 OR 4 . It is synthesized from phthalic anhydride and thymol.

This pH indicator has very peculiar properties, since at extremely low pH it is intense red (pH <0). Subsequently there is a wide pH range where it is completely colorless (pH 1 – 9.2), with a turning range between 9.3 to 10.5 where it can change to blue, while above 10.5 it is blue intense.

Its applications include serving as an acid-base indicator in certain chemical reactions. It is also used as a chromogen in some colorimetric reactions where enzymatic activity is evaluated. For example, in the determination of alkaline phosphatase.

Regarding toxicity, it is less harmful than other pH indicators, being classified by the National Fire Protection Association (NFPA) as level 1 in relation to its effects on health; which means that it is not carcinogenic or mutagenic.

It also does not have a specific toxicity against any target organ; that is, it represents a low risk of aggression to health. However, direct contact with skin and mucosa may cause slight irritation.

On the other hand, in terms of flammability it is classified as 1 and in terms of reactivity as zero.


The pH indicator thymolphthalein in its solid form is a white powder, in its liquid state it is intense red at pH less than zero, colorless at pH 1-9.2 and blue above 10.5. Its molar mass is 430.54 g / mol, with a melting point of (258 ° C), while the boiling point ranges between 248-252 ° C. Density is 0.92 g / mL at 25 ° C

It is insoluble in cold water and soluble in ethanol. It is not an explosive substance.

As a precaution, during transport, storage and handling should be kept away from strong oxidants.


The pH indicator thymolphthalein at 0.1% concentration is generally used for volumetric analysis. To do this, proceed as follows:

– Weigh 100 mg of thymolphthalein and dissolve in 100 ml of 95% ethyl alcohol.

It can also be prepared as follows:

– Weigh 100 mg of thymolphthalein and dissolve in 60 ml of absolute alcohol, and add 40 ml of distilled water.

– Store at room temperature (15 – 25 ° C). It is important that the storage place is cool, ventilated and free from moisture.


Determination of alkaline phosphatase

In determining this test there is a technique that is based on the hydrolytic action that alkaline phosphatase has on thymolphthalein monophosphate. If alkaline phosphatase is present, it will hydrolyze thymolphthalein monophosphate, releasing timolthalein.

This produces a blue tint at alkaline pH. The enzymatic activity of alkaline phosphatase is directly proportional to the intensity of the color formed. The absorbance is read at 590 nm.

Determination of prostate antigen (PSA)

In this sense, Shao F and collaborators in 2018 developed a colorimetric technique in which they use mesoporous silica nanoparticles that trap thymolphthalein molecules, which are strongly bound by the presence of phenyltrimethyloxysilane.

In turn, these nanoparticles have been coated with polyethyleneimine (PEI), favoring the binding of the secondary anti-PSA antibody.

On the other hand, the patient sample has been immobilized on a plate; and when the PSA present is in contact with the nanoparticles, it will bind to the antibodies. Nanoparticles that do not bind will be removed.

Subsequently, thymolphthalein will be easily released by adding an alkaline solution, which generates a blue coloration. The degree of color is directly proportional to the concentration of PSA.

Determination of the activity of the enzyme arginine kinase or phosphokinase

Using the pH indicator thymolphthalein, the enzymatic activity of arginine kinase can be determined. The test is based on the following:

The enzyme arginine kinase is a phosphotransferase that participates in the formation of phosphoarginine, as revealed in the following chemical reaction:

If this reaction is carried out against the pH indicator thymolphthalein, it is possible to measure the enzyme activity. This is possible according to the following explanation: the reaction occurs at an alkaline pH, where thymolphthalein will show its blue color.

Enzyme activity is measured by the fading of the color reflected by the decrease in absorbance at 575 nm, because as the enzyme acts, protons (H +) are released, which acidifies the medium.

This causes the thymolphthalein to lose color once it approaches the turning zone. If the pH falls below 9.2, it becomes completely colorless.

Therefore, the intensity of the blue color is inversely proportional to the enzyme activity.


In the human

This compound is less toxic than most of the pH indicators, since no carcinogenic or mutagenic effects are attributed to it until now. However, it is not totally harmless: on contact with the skin and mucous membranes it can cause redness in the areas mentioned.

It can also produce undesirable reactions if accidentally ingested or inhaled.

In case of direct contact with skin and mucosa, the affected area should be washed with plenty of water for at least 15 minutes. And if they observe signs of irritation, they should go to the nearest healthcare center.

That is why the use of safety implements such as a gown, gloves and safety glasses is recommended when handling.

Despite having a low risk of flammability, it is recommended that for prudence you stay away from lighters.

Impact on the environment

Despite being less toxic to human health, it is toxic in aquatic environments, affecting the organisms found there. In addition, it has a high bioaccumulation power, and in the long term it is possible that degradation products harmful to the environment may be formed.

Therefore, it should be avoided that this product reaches surface or underground waters, as it would affect drinking water.

In this sense, the laboratories that use this indicator must dispose of their waste in compliance with the regulations in force in each country.


  1. Pizzani P, Godoy S, León M, Rueda E, Castañeda M, Arias A. Effect of increasing concentrations of Phytic Phosphorus on the activity of phytase and alkaline phosphatase enzymes in the intestinal epithelium of young sheep. Rev. Cient . (Maracaibo). 2008.18 (1): 59-64. Available at:
  2. “Thymolphthalein.” Wikipedia, The Free Encyclopedia . 22 Feb 2019, 04:40 UTC. 19 May 2019, 22:20,
  3. Shao F, Zhang L, Jiao L, Wang X, Miao L, Li H, Zhou F. Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen Amplified by Releasing pH Indicator Molecules Entrapped in Mesoporous Silica Nanoparticles. Anal Chem. 2018; 17; 90 (14): 8673-8679.
  4. Yu Z, Pan J, Zhou HM. A direct continuous pH-spectrophotometric assay for arginine kinase activity. Protein Pept Lett.  2002; 9 (6): 545-52.
  5. Scharlau. Thymolphthalein Safety Data Sheet. 2013.Available at:
  6. Thymolphthalein Safety Data Sheet. MSDS. National University of Heredia, Costa Rica. Available in: Thymolphthalein% 20 (3).
  7. “Arginine kinase.” Wikipedia, The Free Encyclopedia . 10 Jul 2018, 13:49 UTC. 20 May 2019, 03:58

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