Ninhydrin Test- Definition, Principle, Procedure, Result, Uses

Ninhydrin test is a simple and widely used chemical test to detect the presence of amino acids, peptides, proteins and other compounds that contain a free amino group. Amino acids are the building blocks of proteins and are essential for many biological processes. They can be found in various sources such as food, body fluids, tissues, and forensic samples.

The ninhydrin test is based on the reaction of ninhydrin, a yellow crystalline compound, with the amino group of the test sample. When heated in the presence of ninhydrin, the amino group undergoes deamination and decarboxylation, resulting in the formation of a deep blue or purple colored complex known as Ruhemann`s purple. The intensity of the color is proportional to the amount of amino acid present in the sample. The color can be measured by a spectrophotometer to quantify the amino acid concentration.

The ninhydrin test is useful for both qualitative and quantitative analysis of amino acids and other compounds with an amino group. It can also be used to distinguish carbohydrates from amino acids, as carbohydrates do not react with ninhydrin. However, some exceptions exist, such as proline and hydroxyproline, which produce a yellow-orange color instead of purple. Similarly, some proteins with a free amine group like asparagine produce a brown color.

The ninhydrin test is easy to perform and requires only a few reagents and equipment. It is also sensitive and can detect traces of amino acids in various samples. The ninhydrin test has many applications in biochemistry, forensic science, chromatography, and peptide synthesis. In this article, we will discuss the definition, principle, procedure, result, uses and limitations of the ninhydrin test in detail.

The main objectives of the ninhydrin test are:

• To detect the presence of amines and amino groups in the test solution. Amines and amino groups are organic compounds that contain nitrogen atoms bonded to hydrogen or carbon atoms. They are important components of many biological molecules, such as amino acids, proteins, peptides, and nucleic acids. The ninhydrin test can identify these compounds by their reaction with the ninhydrin reagent, which produces a characteristic color change depending on the type of amine or amino group present.
• To quantify the amino acids present in the sample. Amino acids are the building blocks of proteins and peptides, and they have a central carbon atom attached to an amino group, a carboxyl group, a hydrogen atom, and a variable side chain. The ninhydrin test can measure the concentration of amino acids in the sample by comparing the intensity of the color produced with a standard curve. The color intensity is proportional to the amount of amino acids in the solution, and it varies depending on the nature of the side chain.
• To distinguish carbohydrates from amino acids. Carbohydrates are another class of organic molecules that consist of carbon, hydrogen, and oxygen atoms. They are important sources of energy and structural materials for living organisms. Some carbohydrates, such as monosaccharides and disaccharides, have a similar molecular weight and solubility as amino acids, and they may interfere with the ninhydrin test. However, carbohydrates do not react with the ninhydrin reagent, and they do not produce any color change. Therefore, the ninhydrin test can differentiate between carbohydrates and amino acids based on their reactivity with the reagent.

The principle of the ninhydrin test is based on the fact that two molecules of ninhydrin (2, 2- dihydroxyindane-1, 3-dione) react with a free alpha-amino acid to produce a deep purple or blue color known as Ruhemann’s purple. In this reaction, ninhydrin acts as an oxidizing agent and causes the deamination and decarboxylation of the amino acids at an elevated temperature. This reaction is then followed by condensation between the reduced ninhydrin molecules, released ammonia, and the second molecule of ninhydrin. By the end of the reaction, a diketohydrindene complex is formed which has a deep purple color. The intensity of the formed complex is proportional to the concentration of amino acids in the solution. The color intensity, in turn, depends on the type of amino acid present.

The general reaction of ninhydrin with an amino acid can be represented as follows:

However, not all amino acids react in the same way with ninhydrin. Some amino acids like proline and hydroxyproline, which have a secondary amine group instead of a primary one, yield an iminium salt, which is yellow-orange in color. Similarly, proteins with a free amide group like asparagine, react with the ninhydrin reagent to form a brown colored product. Therefore, the ninhydrin test can also be used to distinguish different types of amino acids and proteins based on their color reactions.

To perform the ninhydrin test, you will need the following reagents, materials and equipment:

Reagents

• Ninhydrin reagent: This is the main reagent that reacts with amino acids to produce a colored complex. You can prepare it by dissolving 0.35 g of ninhydrin in 100 ml of ethanol. You can also use iso-propanol or a 1:1 mixture of acetone and butanol instead of ethanol.
• Diluent solvent: This is used to dilute the ninhydrin reagent and the sample solution for the quantitative test. You can prepare it by mixing equal volumes of water and n-propanol.
• Standard solution: This is a known concentration of protein or amino acid solution that is used as a reference for the quantitative test. You can use a 1% protein solution or any other suitable concentration.
• Sample solution: This is the unknown solution that you want to test for the presence and amount of amino acids.

Materials

• Test tubes: You will need several test tubes to hold the solutions and perform the reactions. Make sure they are clean and dry before use.
• Test tube stand: You will need a test tube stand to hold the test tubes in place and prevent them from falling or spilling.
• Pipette: You will need a pipette to measure and transfer accurate volumes of solutions. Use a different pipette for each solution to avoid cross-contamination.

Equipment

• Water bath: You will need a water bath to heat the solutions and accelerate the reaction between ninhydrin and amino acids. Set the temperature to 90°C or boiling point depending on the protocol.
• Spectrophotometer: You will need a spectrophotometer to measure the absorbance of the solutions at a specific wavelength. This will help you determine the concentration of amino acids in the sample solution by comparing it with the standard curve. Use a wavelength of 570 nm for most amino acids and 440 nm for proline and hydroxyproline.

These are the requirements for performing the ninhydrin test. Make sure you have all of them ready before starting the experiment.

The procedure of ninhydrin test varies depending on whether you want to perform a qualitative or a quantitative analysis of the sample.

For qualitative analysis

The qualitative analysis is used to detect the presence or absence of amino acids in the sample. The steps are as follows:

• Take 1 ml of standard protein solution in one test tube and 1 ml of the test sample in another dry test tube.
• Add a few drops of ninhydrin reagent to both the test tubes.
• Place the test tubes in a water bath for 5 minutes and then allow them to cool to room temperature.
• Observe the formation of color and note down the result.

For quantitative analysis

The quantitative analysis is used to measure the concentration of amino acids in the sample. The steps are as follows:

• Pipette out different volumes (10 µl, 20 µl, and so on) of the protein solution from the supplied stock solution into a series of test tubes and make up the volume to 1 mL with distilled water.
• Take a tube labeled as one as blank containing 1ml of just distilled water and the rest of the tubes labeled 2 to 9 for construction of a standard curve. Tubes 10-15 are for the unknown samples.
• Add 1 ml of the ninhydrin reagent and 5 ml of diluent solvent to each tube and mix well by vortexing.
• Cover the tubes with caps on top and incubate at 90°C for 17 minutes or boiling water bath for 20 minutes.
• Cool the tubes to room temperature and measure the optical density of the solutions at 570 nm (440 nm for proline and hydroxyproline) against a blank.
• Prepare a standard curve of absorbance against amino acid concentration.
• Determine the amount of amino acid in the unknown sample by plotting a standard curve of A570 on the Y-axis and concentration of amino acid on the X-axis.

The result of the ninhydrin test depends on the color of the complex formed after the reaction of ninhydrin with the amino group of the analyte. The color can vary depending on the type of amino acid present in the sample. The following table summarizes the possible colors and their interpretations:

The intensity of the color is proportional to the concentration of amino acids in the sample. Therefore, a quantitative analysis can be performed by measuring the absorbance of the solution at a specific wavelength using a spectrophotometer. A standard curve can be plotted by using known concentrations of amino acids and their corresponding absorbances. Then, the unknown concentration can be determined by interpolating from the standard curve .

Ninhydrin test is a versatile and sensitive method that has various applications in different fields of science. Some of the uses of ninhydrin test are:

• Biochemistry: Ninhydrin test is used to detect the presence of amino acids in unknown samples. This test can help identify the type and quantity of amino acids in biological fluids, tissues, or food products. This test is also used in solid-phase peptide synthesis to monitor the protection and deprotection steps for amino acid analysis of proteins. Ninhydrin test can also be used to determine the amino acid composition of peptides and proteins by hydrolyzing them with acid or base and then reacting with ninhydrin.
• Forensics: Ninhydrin test is commonly used to detect fingerprints on porous surfaces such as paper, cardboard, or wood. It is possible because the terminal amines of lysine residues in peptides and proteins shed off in fingerprints react with ninhydrin and produce a purple color. Ninhydrin test can reveal latent fingerprints that are not visible to the naked eye or other methods. Ninhydrin test can also be used to detect other trace evidence such as saliva, urine, or blood on crime scenes.
• Chemistry: Ninhydrin test is used to detect the presence of ammonia, primary/secondary amines, or amino acids in chemical compounds or reactions. This test can help identify the functional groups or intermediates in organic synthesis or analysis. Ninhydrin test can also be used to monitor the progress of reactions involving amines or amino acids by measuring the color intensity or absorbance of the product.

• Ninhydrin test is not specific for amino acids as it can also react with other compounds that have free amino groups, such as ammonia, urea, and some drugs. This can result in false-positive results or interference with the color intensity.
• Ninhydrin test is not sensitive enough to detect low concentrations of amino acids in the sample. The minimum detectable concentration of amino acids by this test is about 10 µg/ml. Therefore, this test may not be suitable for trace analysis of amino acids.
• Ninhydrin test is not applicable for all amino acids as some amino acids do not react with ninhydrin or produce different colors. For example, proline and hydroxyproline produce yellow-orange color instead of purple, and cysteine and cystine do not react with ninhydrin at all. Therefore, this test cannot distinguish between different types of amino acids in the sample.
• Ninhydrin test is affected by the pH and temperature of the reaction. The optimal pH for this test is around 5.0 and the optimal temperature is around 90°C. Deviation from these conditions can affect the reaction rate and the color formation. Therefore, this test requires careful control of the experimental conditions to ensure accuracy and reproducibility.

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