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    Ninhydrin Test - Definition, Reaction, Principle, Result, FAQs

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    Ninhydrin Test - Definition, Reaction, Principle, Result, FAQs

    Edited By Team Careers360 | Updated on Jul 05, 2022 05:34 PM IST

    Definition of the Ninhydrin Test

    The ninhydrin test is a chemical test used to determine the presence of ammonia, primary and secondary amines, or amino acids. In this test, ninhydrin reagent is added to the test sample, resulting in the production of a deep blue colour, also known as Ruhemann's purple, in the presence of an amino group.

    Ninhydrin reagent composition

    Ninhydrin reagent is known as 2,2-dihydroxyindane-1,3 dione.

    Ninhydrin structure

    Ninhydrin

    Define deamination

    Deamination is defined as the removal of alpha amino group from the molecule.

    What exactly is the Ninhydrin Test?

    The ninhydrin test is a chemical test that determines if an analyte contains amines or -amino acids chemical test solution is combined with ninhydrin in this assay (a chemical compound with the formula C9H6O4 and the IUPAC name 2,2-dihydroxybenzene-1,3-dione). The presence of ammonia, primary and secondary amines, or amino acids in the analyte is indicated by the development of a deep blue colour.

    Also read -

    Imino acid example

    The molecule containing both imine group and a carboxylic acid group present in the same carbon atom is referred as imino acid group. Example: proline

    Ninhydrin Test Objectives

    The presence of amines and amino groups in the test solution must be detected.

    To determine the number of amino acids in a sample.

    To differentiate between carbs and amino acids.

    Ninhydrin and its Analogy

    Ninhydrin is the most frequent chemical reagent used to detect latent fingermarks on porous materials such as paper and cardboard. The molecule combines with the amino acid (eccrine) component of the fingerprints deposit to produce Ruhemann's purple, a dark purple result. The chemical processes involved are highly complex, and development parameters such as temperature, acidity (pH), and humidity must be regulated in order to achieve the best outcomes.

    Principle of the Ninhydrin Test

    The amino acid oxidative deamination when exposed to ninhydrin, releasing CO2, NH3, and an aldehyde in order to hydrindantin (which is a reduced form of ninhydrin). The ammonia then reacts with another ninhydrin molecule to form diketo hydrin (also known as the Ruhemann's complex). The ammonia then reacts with another ninhydrin molecule to form diketo hydrin (also referred to as the Ruhemann's complex). The intense blue colour is due to this complex. When Amino-acids such as proline are present in the analyte, a yellow complex is generated. When asparagine is employed, the resultant complex is brown in colour.

    Ninhydrin reaction is shown below.

    Ninhydrin reaction with amino acids

    Ninhydrin test: Principle, Reaction, Reagents, Procedure and Result  Interpretation | Online Biochemistry Notes

    Requirements and reagents

    Ninhydrin reagent Ninhydrin reagent: Dilute 0.35g of ninhydrin in 100 mL of ethanol (isopropanol or a 1:1 mixture of acetone and butanol can be used in place of ethanol).

    Solvent for dilution (for quantitative testing): Combine equal parts water and n-propanol.

    Typical solution (1 percent protein solution)

    Materials needed

    Tubes for testing

    Stand for test tubes

    Pipette

    Equipment

    A bath in water

    Spectrophotometer

    Ninhydrin Test Methodology

    For qualitative research

    In one dry test tube, place 1 ml of standard protein solution and 1 ml of test sample.

    In both test tubes, add a few drops of ninhydrin reagent.

    Place the test tubes in the water bath for 5 minutes before allowing them to cool to room temperature.

    Observe the creation of colour and make a note of the outcome.

    For quantitative research

    Pipette varying volumes (10 l, 20 l, etc.) of the protein solution from the supplied stock solution into a series of test tubes then distil the volume to 1 mL.

    Take a blank tube with 1ml of distilled water and the rest of the tubes labelled 2 to 9 for the building of a standard curve. Tubes 10-15 are for unidentified samples.

    In each tube, add 1 mL of the ninhydrin reagent and 5 mL of the diluent solvent and thoroughly mix by vertexing.

    Allow the tubes to cool.

    Cover the tubes with covers and incubate for 17 minutes at 90°C or 20 minutes in a boiling water bath.

    When measuring the optical density of the solutions against a blank at 570 nm, cool the tubes to ambient temperature (440 nm for proline and hydroxyproline).

    Draw a standard curve of A570 on the Y-axis as well as the concentration of amino acid on the X-axis to quantify the amount of amino acid in the unknown compound.

    The next five steps explain how alpha amino acids combine with Ninhydrin, which is involved in the formation of colour.

    1. alpha-amino acid + ninhydrin ninhydrin reduced +Alpha amino acid +H2O

    This is an oxidative deamination reaction that produces an alpha – amino acid by eliciting two hydrogens from the alpha amino acid. With the creation of a water molecule, the ninhydrin is also reduced and loses an oxygen atom.

    2. alpha-amino acid + H2Oalpha-keto acid + NH3

    Rapid hydrolysis of the NH group inside the alpha-amino acid produces an alpha-keto acid containing an ammonia molecule. This alpha-keto acid was also engaged in the step's decarboxylation reaction.

    3. NH3 + alpha-keto acid aldehyde + CO2

    The overall reaction for the preceding reactions is easily summarised in Reaction (4):

    4. alpha-amino acid + 2 ninhydrins CO2 + aldehyde + final complex (BLUE) + 3H2O

    In summary, ninhydrin, which is initially yellow, combines with amino acids and changes to a deep purple colour. This purple colour is recognised by this method.

    Under heated conditions, to create an aldehyde with one fewer carbon atom than the original amino acid. Along with the aldehyde, a carbon dioxide molecule is formed. These first three processes result in lower ninhydrin and ammonia, which are necessary for colour creation.

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    Ninhydrin Test Results and Interpretation

    Test for Ninhydrin

    The presence of a purple-coloured complex in the tube indicates a good result and the existence of amino acid in the sample.

    The absence of the compound in the tube is a negative outcome, indicating that the sample lacks amino acids.

    We can deduce the unknown concentration samples from the graph.


    Ninhydrin Test

    Ninhydrin Test Applications

    The ninhydrin test detects the presence of amino acids in unknown substances.

    This assay is also used in solid-phase peptide synthesis to check the protection of proteins for amino acid analysis.

    The ninhydrin test is often used to detect fingerprints due to its high sensitivity. It is conceivable because ninhydrin reacts with the terminal amines of lysine residues in peptides and proteins shed off in fingerprints.

    Ninhydrin Test limitations

    Ninhydrin reacts with -amino groups as well as nitrogen in ammonia and other free amines.

    Because steric hindrance prevents ninhydrin from reaching the -amino groups, the Ninhydrin test is ineffective for detecting high molecular weight proteins.

    Also check-

    NCERT Chemistry Notes:

    Frequently Asked Question (FAQs)

    1. Describe how to carry out the Ninhydrin Reaction.

    The purpose of a ninhydrin test is to confirm the presence of an amino group in a given molecule. We will need ninhydrin reagent, test tubes, a carrier solvent such as ethanol or distilled water, a spatula, a warm water bath, and the test sample to perform the ninhydrin reaction. The following are the steps to carry out the reaction:


    Using a carrier solvent such as acetone or ethanol, we make a 2% solution of ninhydrin. We achieve this by dissolving 0.2g of ninhydrin in 10ml of the solvent.


    Then, using distilled water, we make a solution of the indicated test chemical.


    Then, using distilled water, we make a solution of the indicated test chemical.


    We put the test solution in a test tube and add a few drips of the ninhydrin solution to it. We let the test tube settle for a few minutes in a water bath at a slightly increased temperature.


    We have a positive ninhydrin test result if the colour of the solution shifts to a deep blue coloration.

    2. Explain the Ninhydrin Reaction Mechanism.

    The mechanism of the ninhydrin reaction is simply an oxidation and reduction reaction process. When we add droplets of ninhydrin solution to a test sample, the ninhydrin works as an oxidising agent. It interacts with the compound's amino group, causing delamination. This procedure produces two gaseous by-products: ammonia and carbon dioxide. In addition to producing an aldehyde, this redox reaction reduces the ninhydrin and produces a reduced product known as hydrindantin. The ammonia that has been released now interacts with another molecule of ninhydrin to generate a di-ketohydrin complex with a deep blue coloration.

    3. A positive ninhydrin test is what colour?

    blue

    A vivid blue coloration in the solution indicates a positive ninhydrin test. This reaction suggests that amino acids, other amines, and ammonia are present in the test material.

    4. Is ninhydrin carcinogenic?

    It causes irritation to mammalian skin. Various harmful effects of ninhydrin have been described in laboratory animals; nevertheless, no long-term in vivo bioassay has been performed to assess its carcinogenic and co-carcinogenic potential in laboratory animals.

    5. Why does ninhydrin cause a blue stain on the skin?

    Ninhydrin reacts with the skin, causing a blue stain due to the presence of amino acids in the skin. It is capable of detecting ammonia and amines. Because of the presence of amine as a functional group, ninhydrin is also used in protein detection.

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    Production Manager Job Description: A Production Manager is responsible for ensuring smooth running of manufacturing processes in an efficient manner. He or she plans and organises production schedules. The role of Production Manager involves estimation, negotiation on budget and timescales with the clients and managers. 

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    Team Lead

    A Team Leader is a professional responsible for guiding, monitoring and leading the entire group. He or she is responsible for motivating team members by providing a pleasant work environment to them and inspiring positive communication. A Team Leader contributes to the achievement of the organisation’s goals. He or she improves the confidence, product knowledge and communication skills of the team members and empowers them.

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    Quality Systems Manager

    A Quality Systems Manager is a professional responsible for developing strategies, processes, policies, standards and systems concerning the company as well as operations of its supply chain. It includes auditing to ensure compliance. It could also be carried out by a third party. 

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    Merchandiser

    A career as a merchandiser requires one to promote specific products and services of one or different brands, to increase the in-house sales of the store. Merchandising job focuses on enticing the customers to enter the store and hence increasing their chances of buying a product. Although the buyer is the one who selects the lines, it all depends on the merchandiser on how much money a buyer will spend, how many lines will be purchased, and what will be the quantity of those lines. In a career as merchandiser, one is required to closely work with the display staff in order to decide in what way a product would be displayed so that sales can be maximised. In small brands or local retail stores, a merchandiser is responsible for both merchandising and buying. 

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    Procurement Manager

    The procurement Manager is also known as  Purchasing Manager. The role of the Procurement Manager is to source products and services for a company. A Procurement Manager is involved in developing a purchasing strategy, including the company's budget and the supplies as well as the vendors who can provide goods and services to the company. His or her ultimate goal is to bring the right products or services at the right time with cost-effectiveness. 

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    Production Planner

    Individuals who opt for a career as a production planner are professionals who are responsible for ensuring goods manufactured by the employing company are cost-effective and meets quality specifications including ensuring the availability of ready to distribute stock in a timely fashion manner. 

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    ITSM Manager

    ITSM Manager is a professional responsible for heading the ITSM (Information Technology Service Management) or (Information Technology Infrastructure Library) processes. He or she ensures that operation management provides appropriate resource levels for problem resolutions. The ITSM Manager oversees the level of prioritisation for the problems, critical incidents, planned as well as proactive tasks. 

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    Information Security Manager

    Individuals in the information security manager career path involves in overseeing and controlling all aspects of computer security. The IT security manager job description includes planning and carrying out security measures to protect the business data and information from corruption, theft, unauthorised access, and deliberate attack 

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    Computer Programmer

    Careers in computer programming primarily refer to the systematic act of writing code and moreover include wider computer science areas. The word 'programmer' or 'coder' has entered into practice with the growing number of newly self-taught tech enthusiasts. Computer programming careers involve the use of designs created by software developers and engineers and transforming them into commands that can be implemented by computers. These commands result in regular usage of social media sites, word-processing applications and browsers.

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    Product Manager

    A Product Manager is a professional responsible for product planning and marketing. He or she manages the product throughout the Product Life Cycle, gathering and prioritising the product. A product manager job description includes defining the product vision and working closely with team members of other departments to deliver winning products.  

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    IT Consultant

    An IT Consultant is a professional who is also known as a technology consultant. He or she is required to provide consultation to industrial and commercial clients to resolve business and IT problems and acquire optimum growth. An IT consultant can find work by signing up with an IT consultancy firm, or he or she can work on their own as independent contractors and select the projects he or she wants to work on.

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    Data Architect

    A Data Architect role involves formulating the organisational data strategy. It involves data quality, flow of data within the organisation and security of data. The vision of Data Architect provides support to convert business requirements into technical requirements. 

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    Security Engineer

    The Security Engineer is responsible for overseeing and controlling the various aspects of a company's computer security. Individuals in the security engineer jobs include developing and implementing policies and procedures to protect the data and information of the organisation. In this article, we will discuss the security engineer job description, security engineer skills, security engineer course, and security engineer career path.

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    UX Architect

    A UX Architect is someone who influences the design processes and its outcomes. He or she possesses a solid understanding of user research, information architecture, interaction design and content strategy. 

     

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